Anti-IL-TIF antibodies and methods of making

ABSTRACT

The present invention relates to ZCYTO18 polynucleotide and polypeptide molecules. The ZCYTO18 is a novel cytokine. The polypeptides may be used within methods for stimulating the proliferation and/or development of hematopoietic cells in vitro and in vivo. The present invention also includes methods for producing the protein, uses therefor and antibodies thereto.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/806,294, filed on Mar. 22, 2004, now U.S. Pat. No. 7,563,568, whichis a continuation of U.S. patent application Ser. No. 09/746,375, filedon Dec. 22, 2000, now abandoned which claims benefit of U.S. ProvisionalApplication Ser. No. 60/172,105, filed on Dec. 23, 1999, and U.S.Provisional Application Ser. No. 60/250,841, filed on Dec. 1, 2000, allof which are herein incorporated by reference. Under 35 U.S.C.§119(e)(1), this application claims benefit of said ProvisionalApplications.

BACKGROUND OF THE INVENTION

Hormones and polypeptide growth factors control proliferation anddifferentiation of cells of multicellular organisms. These diffusiblemolecules allow cells to communicate with each other and act in concertto form cells and organs, and to repair damaged tissue. Examples ofhormones and growth factors include the steroid hormones (e.g. estrogen,testosterone), parathyroid hormone, follicle stimulating hormone, theinterleukins, platelet derived growth factor (PDGF), epidermal growthfactor (EGF), granulocyte-macrophage colony stimulating factor (GM-CSF),erythropoietin (EPO) and calcitonin.

Hormones and growth factors influence cellular metabolism by binding toreceptors. Receptors may be integral membrane proteins that are linkedto signaling pathways within the cell, such as second messenger systems.Other classes of receptors are soluble molecules, such as the nuclearreceptors or transcription factors.

Cytokines generally stimulate proliferation or differentiation of cellsof the hematopoietic lineage or participate in the immune andinflammatory response mechanisms of the body. Examples of cytokines thataffect hematopoiesis are erythropoietin (EPO), which stimulates thedevelopment of red blood cells; thrombopoietin (TPO), which stimulatesdevelopment of cells of the megakaryocyte lineage; andgranulocyte-colony stimulating factor (G-CSF), which stimulatesdevelopment of neutrophils. These cytokines are useful in restoringnormal blood cell levels in patients suffering from anemia,thrombocytopenia, and neutropenia or receiving chemotherapy for cancer.

The interleukins are a family of cytokines that mediate immunologicalresponses, including inflammation. The interleukins mediate a variety ofinflammatory pathologies. Central to an immune response is the T cell,which produce many cytokines and adaptive immunity to antigens.Cytokines produced by the T cell have been classified as type 1 and type2 (Kelso, A. Immun. Cell Biol. 76:300-317, 1998). Type 1 cytokinesinclude IL-2, IFN-γ, LT-α, and are involved in inflammatory responses,viral immunity, intracellular parasite immunity and allograft rejection.Type 2 cytokines include IL-4, IL-5, IL-6, IL-10 and IL-13, and areinvolved in humoral responses, helminth immunity and allergic response.Shared cytokines between Type 1 and 2 include IL-3, GM-CSF and TNF-α.There is some evidence to suggest that Type 1 and Type 2 producing Tcell populations preferentially migrate into different types of inflamedtissue.

Mature T cells may be activated, i.e., by an antigen or other stimulus,to produce, for example, cytokines, biochemical signaling molecules, orreceptors that further influence the fate of the T cell population.

B cells can be activated via receptors on their cell surface including Bcell receptor and other accessory molecules to perform accessory cellfunctions, such as production of cytokines.

Natural killer (NK) cells have a common progenitor cell with T cells andB cells, and play a role in immune surveillance. NK cells, whichcomprise up 15% of blood lymphocytes, do not express antigen receptors,and therefore do not use MHC recognition as requirement for binding to atarget cell. NK cells are involved in the recognition and killing ofcertain tumor cells and virally infected cells. In vivo, NK cells arebelieved to require activation, however, in vitro, NK cells have beenshown to kill some types of tumor cells without activation.

The demonstrated in vivo activities of the cytokine family illustratesthe enormous clinical potential of, and need for, other cytokines,cytokine agonists, and cytokine antagonists. The present inventionaddresses these needs by providing a new cytokine that stimulatesmultiple cell types including hematopoietic cells, and participates inthe inflammatory response and tumor cell growth, as well as relatedcompositions and methods.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a multiple alignment of the human ZCYTO18 polypeptide(hZCYTO18) (SEQ ID NO:3), and the mouse ZCYTO18 polypeptide (mZCYTO18)(SEQ ID NO:38) of the present invention. The “:” in the FIGURE indicatesamino acids that are identical between the mouse and human sequences,and the “.” in the FIGURE indicates amino acids that are conservedsubstitutions. There is a 78.4% identity between the human and mousesequences over the entire sequence (167 amino acid overlap).

DESCRIPTION OF THE INVENTION

The present invention provides such polypeptides for these and otheruses that should be apparent to those skilled in the art from theteachings herein.

Within one aspect, the present invention provides an isolatedpolynucleotide that encodes a cytokine polypeptide comprising a sequenceof amino acid residues that is at least 90% identical to an amino acidsequence selected from the group consisting of: (a) the amino acidsequence as shown in SEQ ID NO:3 from amino acid number 23 (Pro), toamino acid number 167 (Ile); (b) the amino acid sequence as shown in SEQID NO:3 from amino acid number 1 (Met), to amino acid number 167 (Ile);and (c) the amino acid sequence as shown in SEQ ID NO:2 from amino acidnumber 1 (Met), to amino acid number 179 (Ile); and wherein thepolypeptide produced by the cell induces proliferation of cellsexpressing a receptor for the polypeptide comprising zcytor11 (SEQ IDNO:19) or induces cytotoxicity in K562 cells. In one embodiment, theisolated polynucleotide disclosed above is selected from the groupconsisting of: (a) a polynucleotide sequence as shown in SEQ ID NO:1from nucleotide 123 to nucleotide 557; (b) a polynucleotide sequence asshown in SEQ ID NO:1 from nucleotide 57 to nucleotide 557; and (c) apolynucleotide sequence as shown in SEQ ID NO:1 from nucleotide 21 tonucleotide 557; and (d) a polynucleotide sequence complementary to (a),(b) or (c). In another embodiment, the isolated polynucleotide disclosedabove comprises nucleotide 1 to nucleotide 501 of SEQ ID NO:4. Inanother embodiment, the isolated polynucleotide disclosed above encodesa cytokine polypeptide that comprises a sequence of amino acid residuesselected from the group consisting of: (a) the amino acid sequence asshown in SEQ ID NO:3 from amino acid number 23 (Pro), to amino acidnumber 167 (Ile); (b) the amino acid sequence as shown in SEQ ID NO:3from amino acid number 1 (Met), to amino acid number 167 (Ile); and (c)the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 1(Met), to amino acid number 179 (Ile).

Within a second aspect, the present invention provides an expressionvector comprising the following operably linked elements: atranscription promoter; a DNA segment encoding a cytokine polypeptide asshown in SEQ ID NO:3 from amino acid number 23 (Pro), to amino acidnumber 167 (Ile); and a transcription terminator, wherein the promoteris operably linked to the DNA segment, and the DNA segment is operablylinked to the transcription terminator. In one embodiment, theexpression vector disclosed above further comprises a secretory signalsequence operably linked to the DNA segment.

Within a third aspect, the present invention provides a cultured cellcomprising an expression vector according as disclosed above, whereinthe cell expresses a polypeptide encoded by the DNA segment.

Within a fourth aspect, the present invention provides a DNA constructencoding a fusion protein, the DNA construct comprising: a first DNAsegment encoding a polypeptide comprising a sequence of amino acidresidues selected from the group consisting of: (a) the amino acidsequence as shown in SEQ ID NO:3 from amino acid number 1 (Met), toamino acid number 21 (Ala); (b) the amino acid sequence as shown in SEQID NO:3 from amino acid number 41 (Thr), to amino acid number 53 (Leu);(c) the amino acid sequence as shown in SEQ ID NO:3 from amino acidnumber 80 (Met), to amino acid number 91 (Val); (d) the amino acidsequence as shown in SEQ ID NO:3 from amino acid number 103 (Gln), toamino acid number 116 (Arg); (e) the amino acid sequence as shown in SEQID NO:3 from amino acid number 149 (Ile), to amino acid number 162(Leu); and (f) the amino acid sequence as shown in SEQ ID NO:3 fromamino acid number 23 (Pro), to amino acid number 167 (Ile); and at leastone other DNA segment encoding an additional polypeptide, wherein thefirst and other DNA segments are connected in-frame; and wherein thefirst and other DNA segments encode the fusion protein.

Within another aspect, the present invention provides an expressionvector comprising the following operably linked elements: atranscription promoter; a DNA construct encoding a fusion protein asdisclosed above; and a transcription terminator, wherein the promoter isoperably linked to the DNA construct, and the DNA construct is operablylinked to the transcription terminator.

Within another aspect, the present invention provides a cultured cellcomprising an expression vector as disclosed above, wherein the cellexpresses a polypeptide encoded by the DNA construct.

Within another aspect, the present invention provides a method ofproducing a fusion protein comprising: culturing a cell according asdisclosed above; and isolating the polypeptide produced by the cell.

Within another aspect, the present invention provides an isolatedcytokine polypeptide comprising a sequence of amino acid residues thatis at least 90% identical to an amino acid sequence selected from thegroup consisting of: (a) the amino acid sequence as shown in SEQ ID NO:3from amino acid number 23 (Pro), to amino acid number 167 (Ile); (b) theamino acid sequence as shown in SEQ ID NO:3 from amino acid number 1(Met), to amino acid number 167 (Ile); and (c) the amino acid sequenceas shown in SEQ ID NO:2 from amino acid number 1 (Met), to amino acidnumber 179 (Ile); and wherein the polypeptide produced by the cellinduces proliferation of cells expressing a receptor for the polypeptidecomprising zcytor11 (SEQ ID NO:19) or induces cytotoxicity in K562cells. In one embodiment, the isolated polypeptide disclosed abovecomprises a sequence of amino acid residues selected from the groupconsisting of: (a) the amino acid sequence as shown in SEQ ID NO:3 fromamino acid number 23 (Pro), to amino acid number 167 (Ile); (b) theamino acid sequence as shown in SEQ ID NO:3 from amino acid number 1(Met), to amino acid number 167 (Ile); and (c) the amino acid sequenceas shown in SEQ ID NO:2 from amino acid number 1 (Met), to amino acidnumber 179 (Ile).

Within another aspect, the present invention provides a method ofproducing a cytokine polypeptide comprising: culturing a cell asdisclosed above; and isolating the cytokine polypeptide produced by thecell.

Within another aspect, the present invention provides a method ofproducing an antibody to a polypeptide comprising: inoculating an animalwith a polypeptide selected from the group consisting of: (a) apolypeptide consisting of 30 to 144 amino acids, wherein the polypeptideis identical to a contiguous sequence of amino acids in SEQ ID NO:3 fromamino acid number 23 (Gly) to amino acid number 779 (Thr); (b) apolypeptide as disclosed above; (c) a polypeptide consisting of theamino acid sequence of SEQ ID NO:3 from amino acid number 29 (Arg) toamino acid number 34 (Asn); (d) a polypeptide consisting of the aminoacid sequence of SEQ ID NO:3 from amino acid number 121 (His) to aminoacid number 126 (Asp); (e) a polypeptide consisting of the amino acidsequence of SEQ ID NO:3 from amino acid number 134 (Gln) to amino acidnumber 139 (Thr); (f) a polypeptide consisting of the amino acidsequence of SEQ ID NO:3 from amino acid number 137 (Lys) to amino acidnumber 142 (Lys); (g) a polypeptide consisting of the amino acidsequence of SEQ ID NO:3 from amino acid number 145 (Glu) to amino acidnumber 150 (Lys); (h) a polypeptide consisting of the amino acidsequence of SEQ ID NO:3 from amino acid number 41 (Thr), to amino acidnumber 53 (Leu); (i) a polypeptide consisting of the amino acid sequenceof SEQ ID NO:3 from amino acid number 80 (Met) to amino acid number 91(Val); (j) a polypeptide consisting of the amino acid sequence of SEQ IDNO:3 from amino acid number 103 (Met) to amino acid number 116 (Arg);(k) a polypeptide consisting of the amino acid sequence of SEQ ID NO:3from amino acid number 149 (Ile) to amino acid number 162 (Leu); andwherein the polypeptide elicits an immune response in the animal toproduce the antibody; and isolating the antibody from the animal.

Within another aspect, the present invention provides an antibodyproduced by the method as disclosed above, which binds to a polypeptideof SEQ ID NO:2 or SEQ ID NO:3. In one embodiment, the antibody disclosedabove is a monoclonal antibody. Within another aspect, the presentinvention provides an antibody which specifically binds to a polypeptideas disclosed above.

Within another aspect, the present invention provides a method ofdetecting, in a test sample, the presence of an antagonist of ZCYTO18protein activity, comprising: culturing a cell that is responsive to aZCYTO18-stimulated cellular pathway; and producing a polypeptide by themethod as disclosed above; and exposing the polypeptide to the cell, inthe presence and absence of a test sample; and comparing levels ofresponse to the polypeptide, in the presence and absence of the testsample, by a biological or biochemical assay; and determining from thecomparison, the presence of the antagonist of ZCYTO18 activity in thetest sample.

Within another aspect, the present invention provides a method ofdetecting, in a test sample, the presence of an agonist of ZCYTO18protein activity, comprising: culturing a cell that is responsive to aZCYTO18-stimulated cellular pathway; and adding a test sample; andcomparing levels of response in the presence and absence of the testsample, by a biological or biochemical assay; and determining from thecomparison, the presence of the agonist of ZCYTO18 activity in the testsample.

Within another aspect, the present invention provides a method fordetecting a genetic abnormality in a patient, comprising: obtaining agenetic sample from a patient; producing a first reaction product byincubating the genetic sample with a polynucleotide comprising at least14 contiguous nucleotides of SEQ ID NO:1 or the complement of SEQ IDNO:1, under conditions wherein said polynucleotide will hybridize tocomplementary polynucleotide sequence; visualizing the first reactionproduct; and comparing said first reaction product to a control reactionproduct from a wild type patient, wherein a difference between saidfirst reaction product and said control reaction product is indicativeof a genetic abnormality in the patient.

Within another aspect, the present invention provides a method fordetecting a cancer in a patient, comprising: obtaining a tissue orbiological sample from a patient; incubating the tissue or biologicalsample with an antibody as disclosed above under conditions wherein theantibody binds to its complementary polypeptide in the tissue orbiological sample; visualizing the antibody bound in the tissue orbiological sample; and comparing levels of antibody bound in the tissueor biological sample from the patient to a normal control tissue orbiological sample, wherein an increase or decrease in the level ofantibody bound to the patient tissue or biological sample relative tothe normal control tissue or biological sample is indicative of a cancerin the patient.

Within another aspect, the present invention provides a method fordetecting a cancer in a patient, comprising: obtaining a tissue orbiological sample from a patient; labeling a polynucleotide comprisingat least 14 contiguous nucleotides of SEQ ID NO:1 or the complement ofSEQ ID NO:1; incubating the tissue or biological sample with underconditions wherein the polynucleotide will hybridize to complementarypolynucleotide sequence; visualizing the labeled polynucleotide in thetissue or biological sample; and comparing the level of labeledpolynucleotide hybridization in the tissue or biological sample from thepatient to a normal control tissue or biological sample, wherein anincrease or decrease in the labeled polynucleotide hybridization to thepatient tissue or biological sample relative to the normal controltissue or biological sample is indicative of a cancer in the patient.

Within another aspect, the present invention provides a method ofkilling cancer cells comprising, obtaining ex vivo a tissue orbiological sample containing cancer cells from a patient, or identifyingcancer cells in vivo; producing a polypeptide by the method as disclosedabove; formulating the polypeptide in a pharmaceutically acceptablevehicle; and administering to the patient or exposing the cancer cellsto the polypeptide; wherein the polypeptide kills the cells. In oneembodiment, the method of killing cancer cells is as disclosed above,wherein the polypeptide is further conjugated to a toxin.

Within another aspect, the present invention provides a method ofincreasing platelets in a patient or injured tissue, producing apolypeptide by the method as disclosed above; administering thepolypeptide to the patient or injured tissue in a pharmaceuticallyacceptable vehicle, wherein the polypeptide increases the level pfplatelets in the patient or injured tissue.

Within another aspect, the present invention provides a method fordetecting inflammation in a patient, comprising: obtaining a tissue orbiological sample from a patient; incubating the tissue or biologicalsample with an antibody as disclosed above under conditions wherein theantibody binds to its complementary polypeptide in the tissue orbiological sample; visualizing the antibody bound in the tissue orbiological sample; and comparing levels of antibody bound in the tissueor biological sample from the patient to a normal control tissue orbiological sample, wherein an increase in the level of antibody bound tothe patient tissue or biological sample relative to the normal controltissue or biological sample is indicative of inflammation in thepatient.

Within another aspect, the present invention provides a method fordetecting inflammation in a patient, comprising: obtaining a tissue orbiological sample from a patient; labeling a polynucleotide comprisingat least 14 contiguous nucleotides of SEQ ID NO:1 or the complement ofSEQ ID NO:1; incubating the tissue or biological sample with underconditions wherein the polynucleotide will hybridize to complementarypolynucleotide sequence; visualizing the labeled polynucleotide in thetissue or biological sample; and comparing the level of labeledpolynucleotide hybridization in the tissue or biological sample from thepatient to a normal control tissue or biological sample, wherein anincrease in the labeled polynucleotide hybridization to the patienttissue or biological sample relative to the normal control tissue orbiological sample is indicative of inflammation in the patient.

These and other aspects of the invention will become evident uponreference to the following detailed description of the invention.

Prior to setting forth the invention in detail, it may be helpful to theunderstanding thereof to define the following terms:

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985),substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2: 95-107, 1991. DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹M⁻¹.

The term “complements of a polynucleotide molecule” denotes apolynucleotide molecule having a complementary base sequence and reverseorientation as compared to a reference sequence. For example, thesequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

The term “contig” denotes a polynucleotide that has a contiguous stretchof identical or complementary sequence to another polynucleotide.Contiguous sequences are said to “overlap” a given stretch ofpolynucleotide sequence either in their entirety or along a partialstretch of the polynucleotide.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

The term “expression vector” is used to denote a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of interestoperably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

The term “isolated”, when applied to a polynucleotide, denotes that thepolynucleotide has been removed from its natural genetic milieu and isthus free of other extraneous or unwanted coding sequences, and is in aform suitable for use within genetically engineered protein productionsystems. Such isolated molecules are those that are separated from theirnatural environment and include cDNA and genomic clones. Isolated DNAmolecules of the present invention are free of other genes with whichthey are ordinarily associated, but may include naturally occurring 5′and 3′ untranslated regions such as promoters and terminators. Theidentification of associated regions will be evident to one of ordinaryskill in the art (see for example, Dynan and Tijan, Nature 316:774-78,1985).

An “isolated” polypeptide or protein is a polypeptide or protein that isfound in a condition other than its native environment, such as apartfrom blood and animal tissue. In a preferred form, the isolatedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin. It is preferred to provide thepolypeptides in a highly purified form, i.e. greater than 95% pure, morepreferably greater than 99% pure. When used in this context, the term“isolated” does not exclude the presence of the same polypeptide inalternative physical forms, such as dimers or alternatively glycosylatedor derivatized forms.

The term “operably linked”, when referring to DNA segments, indicatesthat the segments are arranged so that they function in concert fortheir intended purposes, e.g., transcription initiates in the promoterand proceeds through the coding segment to the terminator.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

“Paralogs” are distinct but structurally related proteins made by anorganism. Paralogs are believed to arise through gene duplication. Forexample, α-globin, β-globin, and myoglobin are paralogs of each other.

A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides”.

“Probes and/or primers” as used herein can be RNA or DNA. DNA can beeither cDNA or genomic DNA. Polynucleotide probes and primers are singleor double-stranded DNA or RNA, generally synthetic oligonucleotides, butmay be generated from cloned cDNA or genomic sequences or itscomplements. Analytical probes will generally be at least 20 nucleotidesin length, although somewhat shorter probes (14-17 nucleotides) can beused. PCR primers are at least 5 nucleotides in length, preferably 15 ormore nt, more preferably 20-30 nt. Short polynucleotides can be usedwhen a small region of the gene is targeted for analysis. For grossanalysis of genes, a polynucleotide probe may comprise an entire exon ormore. Probes can be labeled to provide a detectable signal, such as withan enzyme, biotin, a radionuclide, fluorophore, chemiluminescer,paramagnetic particle and the like, which are commercially availablefrom many sources, such as Molecular Probes, Inc., Eugene, Oreg., andAmersham Corp., Arlington Heights, Ill., using techniques that are wellknown in the art.

The term “promoter” is used herein for its art-recognized meaning todenote a portion of a gene containing DNA sequences that provide for thebinding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-peptide structure comprising an extracellular ligand-bindingdomain and an intracellular effector domain that is typically involvedin signal transduction. Binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell. This interactionin turn leads to an alteration in the metabolism of the cell. Metabolicevents that are linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. In general, receptors can be membranebound, cytosolic or nuclear; monomeric (e.g., thyroid stimulatinghormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGFreceptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSFreceptor, erythropoietin receptor and IL-6 receptor).

The term “secretory signal sequence” denotes a DNA sequence that encodesa polypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

The term “splice variant” is used herein to denote alternative forms ofRNA transcribed from a gene. Splice variation arises naturally throughuse of alternative splicing sites within a transcribed RNA molecule, orless commonly between separately transcribed RNA molecules, and mayresult in several mRNAs transcribed from the same gene. Splice variantsmay encode polypeptides having altered amino acid sequence. The termsplice variant is also used herein to denote a protein encoded by asplice variant of an mRNA transcribed from a gene.

Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

All references cited herein are incorporated by reference in theirentirety.

The present invention is based in part upon the discovery of a novel DNAsequence that encodes a protein having the structure of afour-helical-bundle cytokine. Through processes of cloning, andexpression studies described herein, a polynucleotide sequence encodinga novel ligand polypeptide has been identified. This polypeptide ligand,designated ZCYTO18, was isolated from T-cell cDNA library and mixedlymphocyte reaction (MLR) cDNA and is expressed in activated humanperipheral blood cells (hPBCs), which were selected for CD3. CD3 is acell surface marker unique to cells of lymphoid origin, particularly Tcells. Based on Northern and RT-PCR analysis, ZCYTO18 polynucleotidesare expressed in T-cells, activated T- and B-cells, and lymphoid tissue.The human ZCYTO18 nucleotide sequence is represented in SEQ ID NO:1.

Analysis of SEQ ID NO:1 reveals that there are two possible initiationMethionine residues for a ZCYTO18 cytokine polypeptide translatedtherefrom. The two deduced ZCYTO18 polypeptide amino acid sequences areshown in SEQ ID NO:2 (a 179 amino acid polypeptide having the initiatingMet at nucleotide 21 in SEQ ID NO:1) and SEQ ID NO:3 (a 167 amino acidpolypeptide having the initiating Met at nucleotide 57 in SEQ ID NO:1).Although both of these sequences encode a ZCYTO18 polypeptide, based onsimilarity of the ZCYTO18 sequence to IL-10 and other cytokines, and thepresence of a strong signal sequence, SEQ ID NO:3 encodes a fullyfunctional secreted cytokine polypeptide.

Sequence analysis of the deduced amino acid sequence as represented inSEQ ID NO:3 indicates a 167 amino acid polypeptide containing a 22 aminoacid residue secretory signal sequence (amino acid residues 1 (Met) to21 (Ala) of SEQ ID NO:3), and a mature polypeptide of 146 amino acids(amino acid residues 22 (Ala) to 167 (Ile) of SEQ ID NO:3). N-terminalsequence shows that the mature start at residue 22 (Ala) of SEQ ID NO:3or 34 (Ala) of SEQ ID NO:2.

In general, cytokines are predicted to have a four-alpha helixstructure, with the 1^(st) and 4^(th) helices being most important inligand-receptor interactions. The 1^(st) and 4^(th) helices are morehighly conserved among members of the family. Referring to the humanZCYTO18 amino acid sequence shown in SEQ ID NO:3, alignment of humanZCYTO18, human IL-10, human zcyto10 (WO US98/25228), and human HumanMDA7 (Genbank Accession No. Q13007) amino acid sequences suggests thatZCYTO18 helix A is defined by amino acid residues 41 (Thr) to 53 (leu)of SEQ ID NO:3; helix B by amino acid residues 80 (Met) to 91 (Val) ofSEQ ID NO:3; helix C by amino acid residues 103 (Met) to 116 (Arg) ofSEQ ID NO:3; and helix D by amino acid residues 149 (Ile) to 162 Leu) ofSEQ ID NO:3. Structural analysis suggests that the A/B loop is long, theB/C loop is short and the C/D loop is long. This loop structure resultsin an up-up-down-down helical organization. Four cysteine residues areconserved between IL-10 and ZCYTO18 corresponding to amino acid residues8, 28, 77 and 120 of SEQ ID NO:3. Consistent cysteine placement isfurther confirmation of the four-helical-bundle structure.

The corresponding polynucleotides encoding the ZCYTO18 polypeptideregions, domains, motifs, residues and sequences described herein are asshown in SEQ ID NO:1. Moreover, the corresponding ZCYTO18 polypeptideregions, domains, motifs, residues and sequences described herein arealso as shown in SEQ ID NO:2.

Four-helical bundle cytokines are also grouped by the length of theircomponent helices. “Long-helix” form cytokines generally consist ofbetween 24-30 residue helices and include IL-6, ciliary neutrotrophicfactor (CNTF), leukemia inhibitory factor (LIF) and human growth hormone(hGH). “Short-helix” form cytokines generally consist of between 18-21residue helices and include IL-2, IL-4 and GM-CSF. Zcyto18 is believedto be a new member of the short-helix form cytokine group. Studies usingCNTF and IL-6 demonstrated that a CNTF helix can be exchanged for theequivalent helix in IL-6, conferring CTNF-binding properties to thechimera. Thus, it appears that functional domains of four-helicalcytokines determined on the basis of structural homology, irrespectiveof sequence identity, and can maintain functional integrity in a chimera(Kallen et al., J. Biol. Chem. 274:11859-11867, 1999). Using similarmethods, putative regions conferring receptor binding specificity inZCYTO18 comprise the regions of amino acid residues of SEQ ID NO:3 thatinclude: residues 53-60, residues 85-91, and residues 121-140. Theseregions will be useful for preparing chimeric molecules, particularlywith other short-helix form cytokines to determine and modulate receptorbinding specificity.

Subsequent to filing, ZCYTO18 was annotated in the literature as IL-TIF.Moreover, receptors for ZCYTO18 were identified comprising zcytor16 (SEQID NO:32, and SEQ ID NO:33) ((commonly owned PCT InternationalApplication No. PCT/US00/32703, filed on Dec. 1, 2000)), zcytor11 (SEQID NO:18, and SEQ ID NO:19) (Commonly owned U.S. Pat. No. 5,965,704),and CRF2-4 (Genbank Accession No. Z17227). Moreover several ZCYTO18responsive cell lines have been identified (Dumontier et al., J.Immunol. 164:1814-1819, 2000; Dumoutier, L. et al., Proc. Nat'l. Acad.Sci. 97:10144-10149, 2000; Xie MH et al., J. Biol. Chem. 275:31335-31339, 2000; Kotenko SV et al., JBC in press), as well as thosethat express the ZCYTO18 receptor subunit zcytor11. Moreover, commonlyowned zcytor16 receptor was shown to bind ZCYTO18 and antagonize itsactivity (SEQ ID NO:3) (commonly owned PCT International Application No.PCT/US00/32703, filed on Dec. 1, 2000); the mouse IL-TIF (ZCYTO18)sequence is shown in Dumontier et al., J. Immunol. 164:1814-1819, 2000),and was independently cloned, designated, mouse ZCYTO18 herein, and isshown in SEQ ID NO:37 and corresponding plypeptide sequence shown in SEQID NO:38. Moreover, commonly owned zcytor11 (U.S. Pat. No. 5,965,704)and CRF2-4 receptor also bind ZCYTO18 (See, WIPO publication WO00/24758; Dumontier et al., J. Immunol. 164:1814-1819, 2000; Spencer, SDet al., J. Exp. Med. 187:571-578, 1998; Gibbs, VC and Pennica Gene186:97-101, 1997 (CRF2-4 cDNA); Xie, MH et al., J. Biol. Chem. 275:31335-31339, 2000; and Kotenko, SV et al., J. Biol. Chem. manuscript inpress M007837200). Moreover, IL-10β receptor may be involved as areceptor for ZCYTO18, and it is believed to be synonymous with CRF2-4(Dumoutier, L. et al., Proc. Nat'l. Acad. Sci. 97:10144-10149, 2000; LiuY et al, J Immunol. 152; 1821-1829, 1994 (IL-10R cDNA). These receptorsare discussed herein in relation to the uses of ZCTYTO18.

The present invention provides polynucleotide molecules, including DNAand RNA molecules, that encode the ZCYTO18 polypeptides disclosedherein. Those skilled in the art will readily recognize that, in view ofthe degeneracy of the genetic code, considerable sequence variation ispossible among these polynucleotide molecules. SEQ ID NO:4 is adegenerate DNA sequence that encompasses all DNAs that encode theZCYTO18 polypeptide of SEQ ID NO:3. Those skilled in the art willrecognize that the degenerate sequence of SEQ ID NO:4 also provides allRNA sequences encoding SEQ ID NO:3 by substituting U for T. Thus,ZCYTO18 polypeptide-encoding polynucleotides comprising nucleotide 1 or66 to nucleotide 501 of SEQ ID NO:4 and their RNA equivalents arecontemplated by the present invention. Table 1 sets forth the one-lettercodes used within SEQ ID NO:4 to denote degenerate nucleotide positions.“Resolutions” are the nucleotides denoted by a code letter. “Complement”indicates the code for the complementary nucleotide(s). For example, thecode Y denotes either C or T, and its complement R denotes A or G, withA being complementary to T, and G being complementary to C.

TABLE 1 Nucleotide Resolution Complement Resolution A A T T C C G G G GC C T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|GW A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T HA|C|T N A|C|G|T N A|C|G|T

The degenerate codons used in SEQ ID NO:4, encompassing all possiblecodons for a given amino acid, are set forth in Table 2.

TABLE 2 One Amino Letter Degenerate Acid Code Codons Codon Cys C TGC TGTTGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro PCCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGNAsn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CARHis H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AARMet M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTNVal V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGGTGG Ter . TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN

One of ordinary skill in the art will appreciate that some ambiguity isintroduced in determining a degenerate codon, representative of allpossible codons encoding each amino acid. For example, the degeneratecodon for serine (WSN) can, in some circumstances, encode arginine(AGR), and the degenerate codon for arginine (MGN) can, in somecircumstances, encode serine (AGY). A similar relationship existsbetween codons encoding phenylalanine and leucine. Thus, somepolynucleotides encompassed by the degenerate sequence may encodevariant amino acid sequences, but one of ordinary skill in the art caneasily identify such variant sequences by reference to the amino acidsequence of SEQ ID NO:3. Variant sequences can be readily tested forfunctionality as described herein.

One of ordinary skill in the art will also appreciate that differentspecies can exhibit “preferential codon usage.” In general, see,Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980; Haas, et al. Curr.Biol. 6:315-24, 1996; Wain-Hobson, et al., Gene 13:355-64, 1981;Grosjean and Fiers, Gene 18:199-209, 1982; Holm, Nuc. Acids Res.14:3075-87, 1986; Ikemura, J. Mol. Biol. 158:573-97, 1982. As usedherein, the term “preferential codon usage” or “preferential codons” isa term of art referring to protein translation codons that are mostfrequently used in cells of a certain species, thus favoring one or afew representatives of the possible codons encoding each amino acid (SeeTable 2). For example, the amino acid Threonine (Thr) may be encoded byACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonlyused codon; in other species, for example, insect cells, yeast, virusesor bacteria, different Thr codons may be preferential. Preferentialcodons for a particular species can be introduced into thepolynucleotides of the present invention by a variety of methods knownin the art. Introduction of preferential codon sequences intorecombinant DNA can, for example, enhance production of the protein bymaking protein translation more efficient within a particular cell typeor species. Therefore, the degenerate codon sequence disclosed in SEQ IDNO:4 serves as a template for optimizing expression of polynucleotidesin various cell types and species commonly used in the art and disclosedherein. Sequences containing preferential codons can be tested andoptimized for expression in various species, and tested forfunctionality as disclosed herein.

As previously noted, the isolated polynucleotides of the presentinvention include DNA and RNA. Methods for preparing DNA and RNA arewell known in the art. In general, RNA is isolated from a tissue or cellthat produces large amounts of ZCYTO18 RNA. Such tissues and cells areidentified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA77:5201, 1980), reverse transcriptase PCR(RT-PCR) or by screeningconditioned medium from various cell types for activity on target cellsor tissue. Once the activity or RNA producing cell or tissue isidentified, total RNA can be prepared using guanidinium isothiocyanateextraction followed by isolation by centrifugation in a CsCl gradient(Chirgwin et al., Biochemistry 18:52-94, 1979). Poly (A)⁺RNA is preparedfrom total RNA using the method of Aviv and Leder (Proc. Natl. Acad.Sci. USA 69:1408-12, 1972). Complementary DNA (cDNA) is prepared frompoly(A)⁺RNA using known methods. In the alternative, genomic DNA can beisolated. Polynucleotides encoding ZCYTO18 polypeptides are thenidentified and isolated by, for example, hybridization or PCR.

A full-length clone encoding ZCYTO18 can be obtained by conventionalcloning procedures. Complementary DNA (cDNA) clones are preferred,although for some applications (e.g., expression in transgenic animals)it may be preferable to use a genomic clone, or to modify a cDNA cloneto include at least one genomic intron. Methods for preparing cDNA andgenomic clones are well known and within the level of ordinary skill inthe art, and include the use of the sequence disclosed herein, or partsthereof, for probing or priming a library. Expression libraries can beprobed with antibodies to ZCYTO18 fragments, or other specific bindingpartners.

Zcyto18 polynucleotide sequences disclosed herein can also be used asprobes or primers to clone 5′ non-coding regions of a ZCYTO18 gene. Inview of the tissue-specific expression observed for ZCYTO18 by Northernblotting and RT PCR (See, Examples 2 and 3), this gene region isexpected to provide for hematopoietic- and lymphoid-specific expression.Promoter elements from a ZCYTO18 gene could thus be used to direct thetissue-specific expression of heterologous genes in, for example,transgenic animals or patients treated with gene therapy. Cloning of 5′flanking sequences also facilitates production of ZCYTO18 proteins by“gene activation” as disclosed in U.S. Pat. No. 5,641,670. Briefly,expression of an endogenous ZCYTO18 gene in a cell is altered byintroducing into the ZCYTO18 locus a DNA construct comprising at least atargeting sequence, a regulatory sequence, an exon, and an unpairedsplice donor site. The targeting sequence is a ZCYTO18 5′ non-codingsequence that permits homologous recombination of the construct with theendogenous ZCYTO18 locus, whereby the sequences within the constructbecome operably linked with the endogenous ZCYTO18 coding sequence. Inthis way, an endogenous ZCYTO18 promoter can be replaced or supplementedwith other regulatory sequences to provide enhanced, tissue-specific, orotherwise regulated expression.

The present invention further provides counterpart polypeptides andpolynucleotides from other species (orthologs). These species include,but are not limited to mammalian, avian, amphibian, reptile, fish,insect and other vertebrate and invertebrate species. Of particularinterest are ZCYTO18 polypeptides from other mammalian species,including murine, porcine, ovine, bovine, canine, feline, equine, andother primate polypeptides. Orthologs of human ZCYTO18 can be clonedusing information and compositions provided by the present invention incombination with conventional cloning techniques. For example, a cDNAcan be cloned using mRNA obtained from a tissue or cell type thatexpresses ZCYTO18 as disclosed herein. Suitable sources of mRNA can beidentified by probing Northern blots with probes designed from thesequences disclosed herein. A library is then prepared from mRNA of apositive tissue or cell line. A ZCYTO18-encoding cDNA can then beisolated by a variety of methods, such as by probing with a complete orpartial human cDNA or with one or more sets of degenerate probes basedon the disclosed sequences. A cDNA can also be cloned using thepolymerase chain reaction, or PCR (Mullis, U.S. Pat. No. 4,683,202),using primers designed from the representative human ZCYTO18 sequencedisclosed herein. Within an additional method, the cDNA library can beused to transform or transfect host cells, and expression of the cDNA ofinterest can be detected with an antibody to ZCYTO18 polypeptide,binding studies or activity assays. Similar techniques can also beapplied to the isolation of genomic clones. Example 5 shows that aZCYTO18 ortholog is present in mouse genomic DNA.

A polynucleotide sequence for the mouse ortholog of human ZCYTO18 hasbeen identified and is shown in SEQ ID NO:37 and the corresponding aminoacid sequence shown in SEQ ID NO:38. Analysis of the mouse ZCYTO18polypeptide encoded by the DNA sequence of SEQ ID NO:37 revealed an openreading frame encoding 179 amino acids (SEQ ID NO:38) comprising apredicted secretory signal peptide of 33 amino acid residues (residue 1(Met) to residue 33 (Ala) of SEQ ID NO:38), and a mature polypeptide of146 amino acids (residue 34 (Leu) to residue 179 (Val) of SEQ ID NO:38).ZCYTO18 helix A is defined by amino acid residues 53 to 65 of SEQ IDNO:38; helix B by amino acid residues 92 to 103 of SEQ ID NO:38; helix Cby amino acid residues 115 to 124 of SEQ ID NO:38; and helix D by aminoacid residues 161 to 174 of SEQ ID NO:38. Four conserved cysteineresidues in mouse ZCYTO18 are conserved with the human sequencecorresponding to amino acid residues 20, 40, 89; and 132 of SEQ IDNO:38. Moreover, in the mouse sequence alternative starting Methionineresidues exist at positions 8 and 13 as shown in SEQ ID NO:38, but thesignal peptide cleavage after residue 33 (Ala) would still result in the146 amino acid mature sequence as described above. The mature sequencefor the mouse ZCYTO18 begins at Leu₃₄ (as shown in SEQ ID NO:38), whichcorresponds to Ala₂₂ (as shown in SEQ ID NO:3) in the human sequence.There is about 78% identity between the mouse and human sequences overthe entire amino acid sequence corresponding to SEQ ID NO:3 and SEQ IDNO:38. The above percent identities were determined using a FASTAprogram with ktup=1, gap opening penalty=12, gap extension penalty=2,and substitution matrix=BLOSUM62, with other FASTA parameters set asdefault. The corresponding polynucleotides encoding the mouse ZCYTO18polypeptide regions, domains, motifs, residues and sequences describedabove are as shown in SEQ ID NO:37.

Those skilled in the art will recognize that the sequence disclosed inSEQ ID NO:1 represents a single allele of human ZCYTO18 and that allelicvariation and alternative splicing are expected to occur. Allelicvariants of this sequence can be cloned by probing cDNA or genomiclibraries from different individuals according to standard procedures.Allelic variants of the DNA sequence shown in SEQ ID NO:1, includingthose containing silent mutations and those in which mutations result inamino acid sequence changes, are within the scope of the presentinvention, as are proteins which are allelic variants of SEQ ID NO:3.cDNAs generated from alternatively spliced mRNAs, which retain theproperties of the ZCYTO18 polypeptide, are included within the scope ofthe present invention, as are polypeptides encoded by such cDNAs andmRNAs. Allelic variants and splice variants of these sequences can becloned by probing cDNA or genomic libraries from different individualsor tissues according to standard procedures known in the art.

Moreover, the genomic structure of ZCYTO18 is readily determined by oneof skill in the art by comparing the cDNA sequence of SEQ ID NO:1 andthe translated amino acid of SEQ ID NO:3 or SEQ ID NO:2 with the genomicDNA in which the gene is contained (e.g, Genbank Accession No.AC007458). For example, such analysis can be readily done using FASTA asdescribed herein. As such, the intron and exon junctions in this regionof genomic DNA can be determined for the ZCYTO18 gene. Thus, the presentinvention includes the ZCYTO18 gene as located in human genomic DNA.Based on annotation of a fragment of human genomic DNA containing a partof ZCYTO18 genomic DNA (Genbank Accession No. AC007458), ZCYTO18 islocated at the 12q15 region of chromosome 12.

Within preferred embodiments of the invention, isolated ZCYTO18-encodingnucleic acid molecules can hybridize under stringent conditions tonucleic acid molecules having the nucleotide sequence of SEQ ID NO:1, tonucleic acid molecules having the nucleotide sequence of nucleotides 87to 587 of SEQ ID NO:1, or to nucleic acid molecules having a nucleotidesequence complementary to SEQ ID NO:1. In general, stringent conditionsare selected to be about 5° C. lower than the thermal melting point(T_(m)) for the specific sequence at a defined ionic strength and pH.The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe.

A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA and DNA-RNA,can hybridize if the nucleotide sequences have some degree ofcomplementarity. Hybrids can tolerate mismatched base pairs in thedouble helix, but the stability of the hybrid is influenced by thedegree of mismatch. The T_(m) of the mismatched hybrid decreases by 1°C. for every 1-1.5% base pair mismatch. Varying the stringency of thehybridization conditions allows control over the degree of mismatch thatwill be present in the hybrid. The degree of stringency increases as thehybridization temperature increases and the ionic strength of thehybridization buffer decreases. Stringent hybridization conditionsencompass temperatures of about 5-25° C. below the T_(m) of the hybridand a hybridization buffer having up to 1 M Na⁺. Higher degrees ofstringency at lower temperatures can be achieved with the addition offormamide which reduces the T_(m) of the hybrid about 1° C. for each 1%formamide in the buffer solution. Generally, such stringent conditionsinclude temperatures of 20-70° C. and a hybridization buffer containingup to 6×SSC and 0-50% formamide. A higher degree of stringency can beachieved at temperatures of from 40-70° C. with a hybridization bufferhaving up to 4×SSC and from 0-50% formamide. Highly stringent conditionstypically encompass temperatures of 42-70° C. with a hybridizationbuffer having up to 1×SSC and 0-50% formamide. Different degrees ofstringency can be used during hybridization and washing to achievemaximum specific binding to the target sequence. Typically, the washesfollowing hybridization are performed at increasing degrees ofstringency to remove non-hybridized polynucleotide probes fromhybridized complexes.

The above conditions are meant to serve as a guide, and it is wellwithin the abilities of one skilled in the art to adapt these conditionsfor use with a particular polynucleotide hybrid. The T_(m) for aspecific target sequence is the temperature (under defined conditions)at which 50% of the target sequence will hybridize to a perfectlymatched probe sequence. Those conditions which influence the T_(m)include, the size and base pair content of the polynucleotide probe, theionic strength of the hybridization solution, and the presence ofdestabilizing agents in the hybridization solution. Numerous equationsfor calculating T_(m) are known in the art, and are specific for DNA,RNA and DNA-RNA hybrids and polynucleotide probe sequences of varyinglength (see, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition (Cold Spring Harbor Press 1989);Ausubel et al., (eds.), Current Protocols in Molecular Biology (JohnWiley and Sons, Inc. 1987); Berger and Kimmel (eds.), Guide to MolecularCloning Techniques, (Academic Press, Inc. 1987); and Wetmur, Crit. Rev.Biochem. Mol. Biol. 26:227 (1990)). Sequence analysis software such asOLIGO 6.0 (LSR; Long Lake, Minn.) and Primer Premier 4.0 (PremierBiosoft International; Palo Alto, Calif.), as well as sites on theInternet, are available tools for analyzing a given sequence andcalculating T_(m) based on user defined criteria. Such programs can alsoanalyze a given sequence under defined conditions and identify suitableprobe sequences. Typically, hybridization of longer polynucleotidesequences, >50 base pairs, is performed at temperatures of about 20-25°C. below the calculated T_(m). For smaller probes, <50 base pairs,hybridization is typically carried out at the T_(m) or 5-10° C. belowthe calculated T_(m). This allows for the maximum rate of hybridizationfor DNA-DNA and DNA-RNA hybrids.

The length of the polynucleotide sequence influences the rate andstability of hybrid formation. Smaller probe sequences, <50 base pairs,reach equilibrium with complementary sequences rapidly, but may formless stable hybrids. Incubation times of anywhere from minutes to hourscan be used to achieve hybrid formation. Longer probe sequences come toequilibrium more slowly, but form more stable complexes, even at lowertemperatures. In such cases, incubations are allowed to proceedovernight or longer. Generally, incubations are carried out for a periodequal to three times the calculated Cot time. Cot time, the time ittakes for the polynucleotide sequences to reassociate, can be calculatedfor a particular sequence by methods known in the art.

The base pair composition of a polynucleotide sequence will affect thethermal stability of its hybrid complex, thereby influencing the choiceof hybridization temperature and the ionic strength of the hybridizationbuffer. A-T pairs are less stable than G-C pairs in aqueous solutionscontaining sodium chloride. Therefore, the higher the G-C content, themore stable the hybrid. Even distribution of G and C residues within thesequence also contributes positively to hybrid stability. In addition,the base pair composition can be manipulated to alter the T_(m) of agiven sequence. For example, 5-methyldeoxycytidine can be substitutedfor deoxycytidine and 5-bromodeoxuridine can be substituted forthymidine to increase the T_(m), whereas 7-deazzo-2′-deoxyguanosine canbe substituted for guanosine to reduce dependence on T_(m).

The ionic concentration of the hybridization buffer also affects thestability of the hybrid. Hybridization buffers generally containblocking agents such as Denhardt's solution (Sigma Chemical Co., St.Louis, Mo.), denatured salmon sperm DNA, tRNA, milk powders (BLOTTO),heparin or SDS, and a Na⁺ source, such as SSC (1×SSC: 0.15 M sodiumchloride, 15 mM sodium citrate) or SSPE (1×SSPE: 1.8 M NaCl, 10 mMNaH₂PO₄, 1 mM EDTA, pH 7.7). By decreasing the ionic concentration ofthe buffer, the stability of the hybrid is increased. Typically,hybridization buffers contain from between 10 mM-1 M Na⁺. The additionof destabilizing or denaturing agents such as formamide,tetralkylammonium salts, guanidinium cations or thiocyanate cations tothe hybridization solution will alter the T_(m) of a hybrid. Typically,formamide is used at a concentration of up to 50% to allow incubationsto be carried out at more convenient and lower temperatures. Formamidealso acts to reduce non-specific background when using RNA probes.

As an illustration, a nucleic acid molecule encoding a variant ZCYTO18polypeptide can be hybridized with a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:1 (or its complement) at 42° C.overnight in a solution comprising 50% formamide, 5×SSC (1×SSC: 0.15 Msodium chloride and 15 mM sodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt's solution (100×Denhardt's solution: 2% (w/v) Ficoll400, 2% (w/v) polyvinylpyrrolidone, and 2% (w/v) bovine serum albumin),10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA.One of skill in the art can devise variations of these hybridizationconditions. For example, the hybridization mixture can be incubated at ahigher temperature, such as about 65° C., in a solution that does notcontain formamide. Moreover, premixed hybridization solutions areavailable (e.g., EXPRESSHYB Hybridization Solution from CLONTECHLaboratories, Inc.), and hybridization can be performed according to themanufacturer's instructions.

Following hybridization, the nucleic acid molecules can be washed toremove non-hybridized nucleic acid molecules under stringent conditions,or under highly stringent conditions. Typical stringent washingconditions include washing in a solution of 0.5×-2×SSC with 0.1% sodiumdodecyl sulfate (SDS) at 55-65° C. That is, nucleic acid moleculesencoding a variant ZCYTO18 polypeptide hybridize with a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1 (or itscomplement) under stringent washing conditions, in which the washstringency is equivalent to 0.5×-2×SSC with 0.1% SDS at 55-65° C.,including 0.5×SSC with 0.1% SDS at 55° C., or 2×SSC with 0.1% SDS at 65°C. One of skill in the art can readily devise equivalent conditions, forexample, by substituting SSPE for SSC in the wash solution.

Typical highly stringent washing conditions include washing in asolution of 0.1×-0.2×SSC with 0.1% sodium dodecyl sulfate (SDS) at50-65° C. In other words, nucleic acid molecules encoding a variantZCYTO18 polypeptide hybridize with a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:1 (or its complement) under highlystringent washing conditions, in which the wash stringency is equivalentto 0.1×-0.2×SSC with 0.1% SDS at 50-65° C., including 0.1×SSC with 0.1%SDS at 50° C., or 0.2×SSC with 0.1% SDS at 65° C.

The present invention also provides isolated ZCYTO18 polypeptides thathave a substantially similar sequence identity to the polypeptides ofSEQ ID NO:3, or their orthologs. The term “substantially similarsequence identity” is used herein to denote polypeptides comprising atleast 70%, at least 80%, at least 90%, at least 95%, or greater than 95%sequence identity to the sequences shown in SEQ ID NO:3, or theirorthologs. The present invention also includes polypeptides thatcomprise an amino acid sequence having at least 70%, at least 80%, atleast 90%, at least 95% or greater than 95% sequence identity to thesequence of amino acid residues 1 to 167, or 23 to 167 of SEQ ID NO:3;or amino acid residues 1 to 179, or 35 to 179 of SEQ ID NO:2. Thepresent invention further includes nucleic acid molecules that encodesuch polypeptides. Methods for determining percent identity aredescribed below.

The present invention also contemplates variant ZCYTO18 nucleic acidmolecules that can be identified using two criteria: a determination ofthe similarity between the encoded polypeptide with the amino acidsequence of SEQ ID NO:3, and/or a hybridization assay, as describedabove. Such ZCYTO18 variants include nucleic acid molecules: (1) thathybridize with a nucleic acid molecule having the nucleotide sequence ofSEQ ID NO:1 (or its complement) under stringent washing conditions, inwhich the wash stringency is equivalent to 0.5×-2×SSC with 0.1% SDS at55-65° C.; or (2) that encode a polypeptide having at least 70%, atleast 80%, at least 90%, at least 95% or greater than 95% sequenceidentity to the amino acid sequence of SEQ ID NO:3. Alternatively,ZCYTO18 variants can be characterized as nucleic acid molecules: (1)that hybridize with a nucleic acid molecule having the nucleotidesequence of SEQ ID NO:1 (or its complement) under highly stringentwashing conditions, in which the wash stringency is equivalent to0.1×-0.2×SSC with 0.1% SDS at 50-65° C.; and (2) that encode apolypeptide having at least 70%, at least 80%, at least 90%, at least95% or greater than 95% sequence identity to the amino acid sequence ofSEQ ID NO:3.

Percent sequence identity is determined by conventional methods. See,for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992).Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.) asshown in Table 3 (amino acids are indicated by the standard one-lettercodes).

$\frac{{Total}\mspace{14mu}{number}\mspace{14mu}{of}\mspace{14mu}{identical}\mspace{14mu}{matches}}{\begin{bmatrix}{{length}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{longer}\mspace{14mu}{sequence}\mspace{14mu}{plus}\mspace{14mu}{the}} \\{{number}\mspace{14mu}{of}\mspace{14mu}{gaps}\mspace{14mu}{introduced}\mspace{14mu}{into}\mspace{14mu}{the}\mspace{14mu}{longer}} \\{{sequence}\mspace{14mu}{in}{\mspace{11mu}\;}{order}\mspace{14mu}{to}\mspace{14mu}{align}\mspace{14mu}{two}\mspace{14mu}{sequences}}\end{bmatrix}} \times 100$

TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of a putativevariant ZCYTO18. The FASTA algorithm is described by Pearson and Lipman,Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth.Enzmmol. 183:63 (1990).

Briefly, FASTA first characterizes sequence similarity by identifyingregions shared by the query sequence (e.g., SEQ ID NO:3) and a testsequence that have either the highest density of identities (if the ktupvariable is 1) or pairs of identities (if ktup=2), without consideringconservative amino acid substitutions, insertions, or deletions. The tenregions with the highest density of identities are then rescored bycomparing the similarity of all paired amino acids using an amino acidsubstitution matrix, and the ends of the regions are “trimmed” toinclude only those residues that contribute to the highest score. Ifthere are several regions with scores greater than the “cutoff” value(calculated by a predetermined formula based upon the length of thesequence and the ktup value), then the trimmed initial regions areexamined to determine whether the regions can be joined to form anapproximate alignment with gaps. Finally, the highest scoring regions ofthe two amino acid sequences are aligned using a modification of theNeedleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol.48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), which allowsfor amino acid insertions and deletions. Preferred parameters for FASTAanalysis are: ktup=1, gap opening penalty=10, gap extension penalty=1,and substitution matrix=BLOSUM62. These parameters can be introducedinto a FASTA program by modifying the scoring matrix file (“SMATRIX”),as explained in Appendix 2 of Pearson, Meth. Enzmmol. 183:63 (1990).

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom three to six, most preferably three, with other FASTA programparameters set as default.

Variant ZCYTO18 polypeptides or polypeptides with substantially similarsequence identity are characterized as having one or more amino acidsubstitutions, deletions or additions. These changes are preferably of aminor nature, that is conservative amino acid substitutions (see Table4) and other substitutions that do not significantly affect the foldingor activity of the polypeptide; small deletions, typically of one toabout 30 amino acids; and amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue, a small linker peptide of up toabout 20-25 residues, or an affinity tag. The present invention thusincludes polypeptides of from about 110 to 180 amino acid residues thatcomprise a sequence that is at least 70%, preferably at least 90%, andmore preferably 95% or more identical to the corresponding region of SEQID NO:3. Polypeptides comprising affinity tags can further comprise aproteolytic cleavage site between the ZCYTO18 polypeptide and theaffinity tag. Preferred such sites include thrombin cleavage sites andfactor Xa cleavage sites.

TABLE 4 Conservative amino acid substitutions Basic: arginine lysinehistidine Acidic: glutamic acid aspartic acid Polar: glutamineasparagine Hydrophobic: leucine isoleucine valine Aromatic:phenylalanine tryptophan tyrosine Small: glycine alanine serinethreonine methionine

Determination of amino acid residues that comprise regions or domainsthat are critical to maintaining structural integrity can be determined.Within these regions one can determine specific residues that will bemore or less tolerant of change and maintain the overall tertiarystructure of the molecule. Methods for analyzing sequence structureinclude, but are not limited to alignment of multiple sequences withhigh amino acid or nucleotide identity, secondary structurepropensities, binary patterns, complementary packing and buried polarinteractions (Barton, Current Opin. Struct. Biol. 5:372-376, 1995 andCordes et al., Current Opin. Struct. Biol. 6:3-10, 1996). In general,when designing modifications to molecules or identifying specificfragments determination of structure will be accompanied by evaluatingactivity of modified molecules.

Amino acid sequence changes are made in ZCYTO18 polypeptides so as tominimize disruption of higher order structure essential to biologicalactivity. For example, when the ZCYTO18 polypeptide comprises one ormore helices, changes in amino acid residues will be made so as not todisrupt the helix geometry and other components of the molecule wherechanges in conformation abate some critical function, for example, anactive site, or binding of the molecule to its binding partners. Theeffects of amino acid sequence changes can be predicted by, for example,computer modeling as disclosed above or determined by analysis ofcrystal structure (see, e.g., Lapthorn et al., Nat. Struct. Biol.2:266-268, 1995). Other techniques that are well known in the artcompare folding of a variant protein to a standard molecule (e.g., thenative protein). For example, comparison of the cysteine pattern in avariant and standard molecules can be made. Mass spectrometry andchemical modification using reduction and alkylation provide methods fordetermining cysteine residues which are associated with disulfide bondsor are free of such associations (Bean et al., Anal. Biochem.201:216-226, 1992; Gray, Protein Sci. 2:1732-1748, 1993; and Pattersonet al., Anal. Chem. 66:3727-3732, 1994). It is generally believed thatif a modified molecule does not have the same cysteine pattern as thestandard molecule folding would be affected. Another well known andaccepted method for measuring folding is circular dichrosism (CD).Measuring and comparing the CD spectra generated by a modified moleculeand standard molecule is routine (Johnson, Proteins 7:205-214, 1990).Crystallography is another well known method for analyzing folding andstructure. Nuclear magnetic resonance (NMR), digestive peptide mappingand epitope mapping are also known methods for analyzing folding andstructurally similarities between proteins and polypeptides (Schaanan etal., Science 257:961-964, 1992).

A Hopp/Woods hydrophilicity profile of the ZCYTO18 protein sequence asshown in SEQ ID NO:3 can be generated (Hopp et al., Proc. Natl. Acad.Sci. 78:3824-3828, 1981; Hopp, J. Immun. Meth. 88:1-18, 1986 andTriquier et al., Protein Engineering 11:153-169, 1998). The profile isbased on a sliding six-residue window. Buried G, S, and T residues andexposed H, Y, and W residues were ignored. For example, in ZCYTO18,hydrophilic regions include: (1) amino acid number 29 (Arg) to aminoacid number 34 (Asn) of SEQ ID NO:3; (2) amino acid number 121 (His) toamino acid number 126 (Asp) of SEQ ID NO:3; (3) amino acid number 134(Gln) to amino acid number 139 (Thr) of SEQ ID NO:3; (4) amino acidnumber 137 (Lys) to amino acid number 142 (Lys) of SEQ ID NO:3; and (5)amino acid number 145 (Glu) to amino acid number 150 (Lys) of SEQ IDNO:2.

Those skilled in the art will recognize that hydrophilicity orhydrophobicity will be taken into account when designing modificationsin the amino acid sequence of a ZCYTO18 polypeptide, so as not todisrupt the overall structural and biological profile. Of particularinterest for replacement are hydrophobic residues selected from thegroup consisting of Val, Leu and Ile or the group consisting of Met,Gly, Ser, Ala, Tyr and Trp. For example, residues tolerant ofsubstitution could include such residues as shown in SEQ ID NO:3.Cysteine residues at positions 8, 27, 77 and 120 of SEQ ID NO:3, will berelatively intolerant of substitution.

The identities of essential amino acids can also be inferred fromanalysis of sequence similarity between IL-10, zcyto10, and MDA7 withZCYTO18. Using methods such as “FASTA” analysis described previously,regions of high similarity are identified within a family of proteinsand used to analyze amino acid sequence for conserved regions. Analternative approach to identifying a variant ZCYTO18 polynucleotide onthe basis of structure is to determine whether a nucleic acid moleculeencoding a potential variant ZCYTO18 gene can hybridize to a nucleicacid molecule having the nucleotide sequence of SEQ ID NO:1, asdiscussed above.

Other methods of identifying essential amino acids in the polypeptidesof the present invention are procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244:1081 (1989), Bass et al., Proc. Natl. Acad. Sci.USA 88:4498 (1991), Coombs and Corey, “Site-Directed Mutagenesis andProtein Engineering,” in Proteins: Analysis and Design, Angeletti (ed.),pages 259-311 (Academic Press, Inc. 1998)). In the latter technique,single alanine mutations are introduced at every residue in themolecule, and the resultant mutant molecules are tested for biologicalactivity as disclosed below to identify amino acid residues that arecritical to the activity of the molecule. See also, Hilton et al., J.Biol. Chem. 271:4699 (1996).

The present invention also includes functional fragments of ZCYTO18polypeptides and nucleic acid molecules encoding such functionalfragments. A “functional” ZCYTO18 or fragment thereof as defined hereinis characterized by its proliferative or differentiating activity, byits ability to induce or inhibit specialized cell functions, or by itsability to bind specifically to an anti-ZCYTO18 antibody, cell, orZCYTO18 receptor (either soluble or immobilized). As previouslydescribed herein, ZCYTO18 is characterized by a four-helical-bundlestructure comprising helix A (amino acid residues 41-53), helix B (aminoacid residues 80-91), helix C (amino acid residues 103-116) and helix D(amino acid residues 149-162), as shown in SEQ ID NO:3. Thus, thepresent invention further provides fusion proteins encompassing: (a)polypeptide molecules comprising one or more of the helices describedabove; and (b) functional fragments comprising one or more of thesehelices. The other polypeptide portion of the fusion protein may becontributed by another four-helical-bundle cytokine, such as IL-10,zcyto10, MDA7, IL-15, IL-2, IL-4 and GM-CSF, or by a non-native and/oran unrelated secretory signal peptide that facilitates secretion of thefusion protein.

Routine deletion analyses of nucleic acid molecules can be performed toobtain functional fragments of a nucleic acid molecule that encodes aZCYTO18 polypeptide. As an illustration, DNA molecules having thenucleotide sequence of SEQ ID NO:1 or fragments thereof, can be digestedwith Bal31 nuclease to obtain a series of nested deletions. These DNAfragments are then inserted into expression vectors in proper readingframe, and the expressed polypeptides are isolated and tested forZCYTO18 activity, or for the ability to bind anti-ZCYTO18 antibodies orZCYTO18 receptor. One alternative to exonuclease digestion is to useoligonucleotide-directed mutagenesis to introduce deletions or stopcodons to specify production of a desired ZCYTO18 fragment.Alternatively, particular fragments of a ZCYTO18 gene can be synthesizedusing the polymerase chain reaction.

Standard methods for identifying functional domains are well-known tothose of skill in the art. For example, studies on the truncation ateither or both termini of interferons have been summarized byHorisberger and Di Marco, Pharmac. Ther. 66:507 (1995). Moreover,standard techniques for functional analysis of proteins are describedby, for example, Treuter et al., Molec. Gen. Genet. 240:113 (1993);Content et al., “Expression and preliminary deletion analysis of the 42kDa 2-5A synthetase induced by human interferon,” in BiologicalInterferon Systems, Proceedings of ISIR-TNO Meeting on InterferonSystems, Cantell (ed.), pages 65-72 (Nijhoff 1987); Herschman, “The EGFReceptor,” in Control of Animal Cell Proliferation 1 Boynton et al.,(eds.) pages 169-199 (Academic Press 1985); Coumailleau et al., J. Biol.Chem. 270:29270 (1995); Fukunaga et al., J. Biol. Chem. 270:25291(1995); Yamaguchi et al., Biochem. Pharmacol. 50:1295 (1995); and Meiselet al., Plant Molec. Biol. 30:1 (1996).

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53 (1988)) or Bowie and Sauer(Proc. Nat'l Acad. Sci. USA 86:2152 (1989)). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832 (1991), Ladner etal., U.S. Pat. No. 5,223,409, Huse, international publication No. WO92/06204), and region-directed mutagenesis (Derbyshire et al., Gene46:145 (1986), and Ner et al., DNA 7:127, (1988)).

Variants of the disclosed ZCYTO18 nucleotide and polypeptide sequencescan also be generated through DNA shuffling as disclosed by Stemmer,Nature 370:389 (1994), Stemmer, Proc. Natl. Acad. Sci. USA 91:10747(1994), and international publication No. WO 97/20078. Briefly, variantDNA molecules are generated by in vitro homologous recombination byrandom fragmentation of a parent DNA followed by reassembly using PCR,resulting in randomly introduced point mutations. This technique can bemodified by using a family of parent DNA molecules, such as allelicvariants or DNA molecules from different species, to introduceadditional variability into the process. Selection or screening for thedesired activity, followed by additional iterations of mutagenesis andassay provides for rapid “evolution” of sequences by selecting fordesirable mutations while simultaneously selecting against detrimentalchanges.

Mutagenesis methods as disclosed herein can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode biologically active polypeptides, or polypeptidesthat bind with anti-ZCYTO18 antibodies or soluble ZCYTO18 receptor, canbe recovered from the host cells and rapidly sequenced using modernequipment. These methods allow the rapid determination of the importanceof individual amino acid residues in a polypeptide of interest, and canbe applied to polypeptides of unknown structure.

In addition, the proteins of the present invention (or polypeptidefragments thereof) can be joined to other bioactive molecules,particularly other cytokines, to provide multi-functional molecules. Forexample, one or more helices from ZCYTO18 can be joined to othercytokines to enhance their biological properties or efficiency ofproduction.

The present invention thus provides a series of novel, hybrid moleculesin which a segment comprising one or more of the helices of ZCYTO18 isfused to another polypeptide. Fusion is preferably done by splicing atthe DNA level to allow expression of chimeric molecules in recombinantproduction systems. The resultant molecules are then assayed for suchproperties as improved solubility, improved stability, prolongedclearance half-life, improved expression and secretion levels, andpharmacodynamics. Such hybrid molecules may further comprise additionalamino acid residues (e.g. a polypeptide linker) between the componentproteins or polypeptides.

Non-naturally occurring amino acids include, without limitation,trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline,trans-4-hydroxyproline, N-methylglycine, allo-threonine,methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine,nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylicacid, dehydroproline, 3- and 4-methylproline, 3,3-dimethylproline,tent-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, and 4-fluorophenylalanine. Several methods are knownin the art for incorporating non-naturally occurring amino acid residuesinto proteins. For example, an in vitro system can be employed whereinnonsense mutations are suppressed using chemically aminoacylatedsuppressor tRNAs. Methods for synthesizing amino acids andaminoacylating tRNA are known in the art. Transcription and translationof plasmids containing nonsense mutations is typically carried out in acell-free system comprising an E. coli S30 extract and commerciallyavailable enzymes and other reagents. Proteins are purified bychromatography. See, for example, Robertson et al., J. Am. Chem. Soc.113:2722 (1991), Ellman et al., Methods Enzymol. 202:301 (1991), Chunget al., Science 259:806 (1993), and Chung et al., Proc. Nat'l Acad. Sci.USA 90:10145 (1993).

In a second method, translation is carried out in Xenopus oocytes bymicroinjection of mutated mRNA and chemically aminoacylated suppressortRNAs (Turcatti et al., J. Biol. Chem. 271:19991 (1996)). Within a thirdmethod, E. coli cells are cultured in the absence of a natural aminoacid that is to be replaced (e.g., phenylalanine) and in the presence ofthe desired non-naturally occurring amino acid(s) (e.g.,2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or4-fluorophenylalanine). The non-naturally occurring amino acid isincorporated into the protein in place of its natural counterpart. See,Koide et al., Biochem. 33:7470 (1994). Naturally occurring amino acidresidues can be converted to non-naturally occurring species by in vitrochemical modification. Chemical modification can be combined withsite-directed mutagenesis to further expand the range of substitutions(Wynn and Richards, Protein Sci. 2:395 (1993)).

A limited number of non-conservative amino acids, amino acids that arenot encoded by the genetic code, non-naturally occurring amino acids,and unnatural amino acids may be substituted for ZCYTO18 amino acidresidues.

The present invention also provides polypeptide fragments or peptidescomprising an epitope-bearing portion of a ZCYTO18 polypeptide describedherein. Such fragments or peptides may comprise an “immunogenicepitope,” which is a part of a protein that elicits an antibody responsewhen the entire protein is used as an immunogen. Immunogenicepitope-bearing peptides can be identified using standard methods (see,for example, Geysen et al., Proc. Nat'l Acad. Sci. USA 81:3998 (1983)).

In contrast, polypeptide fragments or peptides may comprise an“antigenic epitope,” which is a region of a protein molecule to which anantibody can specifically bind. Certain epitopes consist of a linear orcontiguous stretch of amino acids, and the antigenicity of such anepitope is not disrupted by denaturing agents. It is known in the artthat relatively short synthetic peptides that can mimic epitopes of aprotein can be used to stimulate the production of antibodies againstthe protein (see, for example, Sutcliffe et al., Science 219:660(1983)). Accordingly, antigenic epitope-bearing peptides andpolypeptides of the present invention are useful to raise antibodiesthat bind with the polypeptides described herein. Hopp/Woodshydrophilicity profiles can be used to determine regions that have themost antigenic potential (Hopp et al., 1981, ibid. and Hopp, 1986,ibid.). In ZCYTO18 these regions include: (1) amino acid number 29 (Arg)to amino acid number 34 (Asn) of SEQ ID NO:3; (2) amino acid number 121(His) to amino acid number 126 (Asp) of SEQ ID NO:3; (3) amino acidnumber 134 (Gln) to amino acid number 139 (Thr) of SEQ ID NO:3; (4)amino acid number 137 (Lys) to amino acid number 142 (Lys) of SEQ IDNO:3; and (5) amino acid number 145 (Glu) to amino acid number 150 (Lys)of SEQ ID NO:2. Moreover, ZCYTO18 antigenic epitopes as predicted by aJameson-Wolf plot, e.g., using DNASTAR Protean program (DNASTAR, Inc.,Madison, Wis.) serve as preferred antigens, and are readily determinedby one of skill in the art.

Antigenic epitope-bearing peptides and polypeptides preferably containat least four to ten amino acids, at least ten to fifteen amino acids,or about 15 to about 30 amino acids of SEQ ID NO:3. Such epitope-bearingpeptides and polypeptides can be produced by fragmenting a ZCYTO18polypeptide, or by chemical peptide synthesis, as described herein.Moreover, epitopes can be selected by phage display of random peptidelibraries (see, for example, Lane and Stephen, Curr. Opin. Immunol 5:268(1993); and Cortese et al., Curr. Opin. Biotechnol. 7:616 (1996)).Standard methods for identifying epitopes and producing antibodies fromsmall peptides that comprise an epitope are described, for example, byMole, “Epitope Mapping,” in Methods in Molecular Biology, Vol. 10,Manson (ed.), pages 105-116 (The Humana Press, Inc. 1992); Price,“Production and Characterization of Synthetic Peptide-DerivedAntibodies,” in Monoclonal Antibodies: Production, Engineering, andClinical Application, Ritter and Ladyman (eds.), pages 60-84 (CambridgeUniversity Press 1995), and Coligan et al. (eds.), Current Protocols inImmunology, pages 9.3.1-9.3.5 and pages 9.4.1-9.4.11 (John Wiley & Sons1997).

Regardless of the particular nucleotide sequence of a variant ZCYTO18polynucleotide, the polynucleotide encodes a polypeptide that ischaracterized by its proliferative or differentiating activity, itsability to induce or inhibit specialized cell functions, or by theability to bind specifically to an anti-ZCYTO18 antibody or ZCYTO18receptor. More specifically, variant ZCYTO18 polynucleotides will encodepolypeptides which exhibit at least 50% and preferably, greater than70%, 80% or 90%, of the activity of the polypeptide as shown in SEQ IDNO:3.

For any ZCYTO18 polypeptide, including variants and fusion proteins, oneof ordinary skill in the art can readily generate a fully degeneratepolynucleotide sequence encoding that variant using the information setforth in Tables 1 and 2 above.

The present invention further provides a variety of other polypeptidefusions (and related multimeric proteins comprising one or morepolypeptide fusions). For example, a ZCYTO18 polypeptide can be preparedas a fusion to a dimerizing protein as disclosed in U.S. Pat. Nos.5,155,027 and 5,567,584. Preferred dimerizing proteins in this regardinclude immunoglobulin constant region domains. Immunoglobulin-ZCYTO18polypeptide fusions can be expressed in genetically engineered cells (toproduce a variety of multimeric ZCYTO18 analogs). Auxiliary domains canbe fused to ZCYTO18 polypeptides to target them to specific cells,tissues, or macromolecules. For example, a ZCYTO18 polypeptide orprotein could be targeted to a predetermined cell type by fusing aZCYTO18 polypeptide to a ligand that specifically binds to a receptor onthe surface of that target cell. In this way, polypeptides and proteinscan be targeted for therapeutic or diagnostic purposes. A ZCYTO18polypeptide can be fused to two or more moieties, such as an affinitytag for purification and a targeting domain. Polypeptide fusions canalso comprise one or more cleavage sites, particularly between domains.See, Tuan et al., Connective Tissue Research 34:1-9, 1996.

Using the methods discussed herein, one of ordinary skill in the art canidentify and/or prepare a variety of polypeptides that havesubstantially similar sequence identity to amino acid residues 1-167 or23-167 of SEQ ID NO:3, or functional fragments and fusions thereof,wherein such polypeptides or fragments or fusions retain the propertiesof the wild-type protein such as the ability to stimulate proliferation,differentiation, induce specialized cell function or bind the ZCYTO18receptor or ZCYTO18 antibodies.

The ZCYTO18 polypeptides of the present invention, including full-lengthpolypeptides, functional fragments, and fusion polypeptides, can beproduced in genetically engineered host cells according to conventionaltechniques. Suitable host cells are those cell types that can betransformed or transfected with exogenous DNA and grown in culture, andinclude bacteria, fungal cells, and cultured higher eukaryotic cells.Eukaryotic cells, particularly cultured cells of multicellularorganisms, are preferred. Techniques for manipulating cloned DNAmolecules and introducing exogenous DNA into a variety of host cells aredisclosed by Sambrook et al., Molecular Cloning: A Laboratory Manual,2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989, and Ausubel et al., eds., Current Protocols in Molecular Biology,John Wiley and Sons, Inc., NY, 1987.

In general, a DNA sequence encoding a ZCYTO18 polypeptide is operablylinked to other genetic elements required for its expression, generallyincluding a transcription promoter and terminator, within an expressionvector. The vector will also commonly contain one or more selectablemarkers and one or more origins of replication, although those skilledin the art will recognize that within certain systems selectable markersmay be provided on separate vectors, and replication of the exogenousDNA may be provided by integration into the host cell genome. Selectionof promoters, terminators, selectable markers, vectors and otherelements is a matter of routine design within the level of ordinaryskill in the art. Many such elements are described in the literature andare available through commercial suppliers.

To direct a ZCYTO18 polypeptide into the secretory pathway of a hostcell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be that of ZCYTO18, or may be derivedfrom another secreted protein (e.g., t-PA) or synthesized de novo. Thesecretory signal sequence is operably linked to the ZCYTO18 DNAsequence, i.e., the two sequences are joined in the correct readingframe and positioned to direct the newly synthesized polypeptide intothe secretory pathway of the host cell. Secretory signal sequences arecommonly positioned 5′ to the DNA sequence encoding the polypeptide ofinterest, although certain secretory signal sequences may be positionedelsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S.Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830).

Alternatively, the secretory signal sequence contained in thepolypeptides of the present invention is used to direct otherpolypeptides into the secretory pathway. The present invention providesfor such fusion polypeptides. A signal fusion polypeptide can be madewherein a secretory signal sequence comprising amino acid residue 1(Met) to 21 (Ala) of SEQ ID NO:3 is be operably linked to a DNA sequenceencoding another polypeptide using methods known in the art anddisclosed herein. The secretory signal sequence contained in the fusionpolypeptides of the present invention is preferably fusedamino-terminally to an additional peptide to direct the additionalpeptide into the secretory pathway. Such constructs have numerousapplications known in the art. For example, these novel secretory signalsequence fusion constructs can direct the secretion of an activecomponent of a normally non-secreted protein. Such fusions may be usedin vivo or in vitro to direct peptides through the secretory pathway.

Cultured mammalian cells are suitable hosts within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-5, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., ibid.), and liposome-mediated transfection(Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80,1993, and viral vectors (Miller and Rosman, BioTechniques 7:980-90,1989; Wang and Finer, Nature Med. 2:714-6, 1996). The production ofrecombinant polypeptides in cultured mammalian cells is disclosed, forexample, by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S.Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; andRingold, U.S. Pat. No. 4,656,134. Suitable cultured mammalian cellsinclude the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK(ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamsterovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitablecell lines are known in the art and available from public depositoriessuch as the American Type Culture Collection, Manassas, Va. In general,strong transcription promoters are preferred, such as promoters fromSV-40 or cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Othersuitable promoters include those from metallothionein genes (U.S. Pat.Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.

Drug selection is generally used to select for cultured mammalian cellsinto which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems canalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g. hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used. Alternative markers that introducean altered phenotype, such as green fluorescent protein, or cell surfaceproteins such as CD4, CD8, Class I MHC, placental alkaline phosphatasemay be used to sort transfected cells from untransfected cells by suchmeans as FACS sorting or magnetic bead separation technology.

Other higher eukaryotic cells can also be used as hosts, including plantcells, insect cells and avian cells. The use of Agrobacterium rhizogenesas a vector for expressing genes in plant cells has been reviewed bySinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987. Transformation ofinsect cells and production of foreign polypeptides therein is disclosedby Guarino et al., U.S. Pat. No. 5,162,222 and WIPO publication WO94/06463. Insect cells can be infected with recombinant baculovirus,commonly derived from Autographa californica nuclear polyhedrosis virus(AcNPV). See, King, L. A. and Possee, R. D., The Baculovirus ExpressionSystem: A Laboratory Guide, London, Chapman & Hall; O'Reilly, D. R. etal., Baculovirus Expression Vectors: A Laboratory Manual, New York,Oxford University Press., 1994; and, Richardson, C. D., Ed., BaculovirusExpression Protocols. Methods in Molecular Biology, Totowa, N. J.,Humana Press, 1995. The second method of making recombinant baculovirusutilizes a transposon-based system described by Luckow (Luckow, V. A, etal., J Virol 67:4566-79, 1993). This system is sold in the Bac-to-Backit (Life Technologies, Rockville, Md.). This system utilizes a transfervector, pFastBac1™ (Life Technologies) containing a Tn7 transposon tomove the DNA encoding the ZCYTO18 polypeptide into a baculovirus genomemaintained in E. coli as a large plasmid called a “bacmid.” ThepFastBac1™ transfer vector utilizes the AcNPV polyhedrin promoter todrive the expression of the gene of interest, in this case ZCYTO18.However, pFastBac 1™ can be modified to a considerable degree. Thepolyhedrin promoter can be removed and substituted with the baculovirusbasic protein promoter (also known as Pcor, p6.9 or MP promoter) whichis expressed earlier in the baculovirus infection, and has been shown tobe advantageous for expressing secreted proteins. See, Hill-Perkins, M.S. and Possee, R. D., J. Gen. Virol. 71:971-6, 1990; Bonning, B. C. etal., J. Gen. Virol. 75:1551-6, 1994; and, Chazenbalk, G. D., andRapoport, B., J. Biol. Chem. 270:1543-9, 1995. In such transfer vectorconstructs, a short or long version of the basic protein promoter can beused. Moreover, transfer vectors can be constructed which replace thenative ZCYTO18 secretory signal sequences with secretory signalsequences derived from insect proteins. For example, a secretory signalsequence from Ecdysteroid Glucosyltransferase (EGT), honey bee Melittin(Invitrogen, Carlsbad, Calif.), or baculovirus gp67 (PharMingen, SanDiego, Calif.) can be used in constructs to replace the native ZCYTO18secretory signal sequence. In addition, transfer vectors can include anin-frame fusion with DNA encoding an epitope tag at the C- or N-terminusof the expressed ZCYTO18 polypeptide, for example, a Glu-Glu epitope tag(Grussenmeyer, T. et al., Proc. Natl. Acad. Sci. 82:7952-4, 1985). Usingtechniques known in the art, a transfer vector containing ZCYTO18 istransformed into E. Coli, and screened for bacmids which contain aninterrupted lacZ gene indicative of recombinant baculovirus. The bacmidDNA containing the recombinant baculovirus genome is isolated, usingcommon techniques, and used to transfect Spodoptera frugiperda cells,e.g. Sf9 cells. Recombinant virus that expresses ZCYTO18 is subsequentlyproduced. Recombinant viral stocks are made by methods commonly used theart.

The recombinant virus is used to infect host cells, typically a cellline derived from the fall armyworm, Spodoptera frugiperda. See, ingeneral, Glick and Pasternak, Molecular Biotechnology: Principles andApplications of Recombinant DNA, ASM Press, Washington, D.C., 1994.Another suitable cell line is the High FiveO™ cell line (Invitrogen)derived from Trichoplusia ni (U.S. Pat. No. 5,300,435). Commerciallyavailable serum-free media are used to grow and maintain the cells.Suitable media are Sf900 II™ (Life Technologies) or ESF 921™ (ExpressionSystems) for the Sf9 cells; and Ex-cellO405™ (JRH Biosciences, Lenexa,Kans.) or Express FiveO™ (Life Technologies) for the T. ni cells. Thecells are grown up from an inoculation density of approximately 2-5×10⁵cells to a density of 1-2×10⁶ cells at which time a recombinant viralstock is added at a multiplicity of infection (MOI) of 0.1 to 10, moretypically near 3. Procedures used are generally described in availablelaboratory manuals (King, L. A. and Possee, R. D., ibid.; O′Reilly, D.R. et al., ibid.; Richardson, C. D., ibid.). Subsequent purification ofthe ZCYTO18 polypeptide from the supernatant can be achieved usingmethods described herein.

Fungal cells, including yeast cells, can also be used within the presentinvention. Yeast species of particular interest in this regard includeSaccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.Methods for transforming S. cerevisiae cells with exogenous DNA andproducing recombinant polypeptides therefrom are disclosed by, forexample, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat.No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat.No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformedcells are selected by phenotype determined by the selectable marker,commonly drug resistance or the ability to grow in the absence of aparticular nutrient (e.g., leucine). A preferred vector system for usein Saccharomyces cerevisiae is the POT1 vector system disclosed byKawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformedcells to be selected by growth in glucose-containing media. Suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman etal., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446;5,063,154; 5,139,936 and 4,661,454. Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia methanolica, Pichia guillermondii and Candida maltosaare known in the art. See, for example, Gleeson et al., J. Gen.Microbiol. 132:3459-65, 1986 and Cregg, U.S. Pat. No. 4,882,279.Aspergillus cells may be utilized according to the methods of McKnightet al., U.S. Pat. No. 4,935,349. Methods for transforming Acremoniumchrysogenum are disclosed by Sumino et al., U.S. Pat. No. 5,162,228.Methods for transforming Neurospora are disclosed by Lambowitz, U.S.Pat. No. 4,486,533.

The use of Pichia methanolica as host for the production of recombinantproteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO98/02536, and WO 98/02565. DNA molecules for use in transforming P.methanolica will commonly be prepared as double-stranded, circularplasmids, which are preferably linearized prior to transformation. Forpolypeptide production in P. methanolica, it is preferred that thepromoter and terminator in the plasmid be that of a P. methanolica gene,such as a P. methanolica alcohol utilization gene (AUG1 or AUG2). Otheruseful promoters include those of the dihydroxyacetone synthase (DHAS),formate dehydrogenase (FMD), and catalase (CAT) genes. To facilitateintegration of the DNA into the host chromosome, it is preferred to havethe entire expression segment of the plasmid flanked at both ends byhost DNA sequences. A preferred selectable marker for use in Pichiamethanolica is a P. methanolica ADE2 gene, which encodesphosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), whichallows ade2 host cells to grow in the absence of adenine. Forlarge-scale, industrial processes where it is desirable to minimize theuse of methanol, it is preferred to use host cells in which bothmethanol utilization genes (AUG1 and AUG2) are deleted. For productionof secreted proteins, host cells deficient in vacuolar protease genes(PEP4 and PRB1) are preferred. Electroporation is used to facilitate theintroduction of a plasmid containing DNA encoding a polypeptide ofinterest into P. methanolica cells. It is preferred to transform P.methanolica cells by electroporation using an exponentially decaying,pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm,preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40milliseconds, most preferably about 20 milliseconds.

Prokaryotic host cells, including strains of the bacteria Escherichiacoli, Bacillus and other genera are also useful host cells within thepresent invention. Techniques for transforming these hosts andexpressing foreign DNA sequences cloned therein are well known in theart (see, e.g., Sambrook et al., ibid.). When expressing a ZCYTO18polypeptide in bacteria such as E. coli, the polypeptide may be retainedin the cytoplasm, typically as insoluble granules, or may be directed tothe periplasmic space by a bacterial secretion sequence. In the formercase, the cells are lysed, and the granules are recovered and denaturedusing, for example, guanidine isothiocyanate or urea. The denaturedpolypeptide can then be refolded and dimerized by diluting thedenaturant, such as by dialysis against a solution of urea and acombination of reduced and oxidized glutathione, followed by dialysisagainst a buffered saline solution. In the latter case, the polypeptidecan be recovered from the periplasmic space in a soluble and functionalform by disrupting the cells (by, for example, sonication or osmoticshock) to release the contents of the periplasmic space and recoveringthe protein, thereby obviating the need for denaturation and refolding.

Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell. P. methanolicacells are cultured in a medium comprising adequate sources of carbon,nitrogen and trace nutrients at a temperature of about 25° C. to 35° C.Liquid cultures are provided with sufficient aeration by conventionalmeans, such as shaking of small flasks or sparging of fermentors. Apreferred culture medium for P. methanolica is YEPD (2% D-glucose, 2%Bacto™ Peptone (Difco Laboratories, Detroit, Mich.), 1% Bacto™ yeastextract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).

It is preferred to purify the polypeptides of the present invention to≧80% purity, more preferably to ≧90% purity, even more preferably ≧95%purity, and particularly preferred is a pharmaceutically pure state,that is greater than 99.9% pure with respect to contaminatingmacromolecules, particularly other proteins and nucleic acids, and freeof infectious and pyrogenic agents. Preferably, a purified polypeptideis substantially free of other polypeptides, particularly otherpolypeptides of animal origin.

Expressed recombinant ZCYTO18 polypeptides (or chimeric ZCYTO18polypeptides) can be purified using fractionation and/or conventionalpurification methods and media. Ammonium sulfate precipitation and acidor chaotrope extraction may be used for fractionation of samples.Exemplary purification steps may include hydroxyapatite, size exclusion,FPLC and reverse-phase high performance liquid chromatography. Suitablechromatographic media include derivatized dextrans, agarose, cellulose,polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Qderivatives are preferred. Exemplary chromatographic media include thosemedia derivatized with phenyl, butyl, or octyl groups, such asPhenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas,Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like; orpolyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.Suitable solid supports include glass beads, silica-based resins,cellulosic resins, agarose beads, cross-linked agarose beads,polystyrene beads, cross-linked polyacrylamide resins and the like thatare insoluble under the conditions in which they are to be used. Thesesupports may be modified with reactive groups that allow attachment ofproteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxylgroups and/or carbohydrate moieties. Examples of coupling chemistriesinclude cyanogen bromide activation, N-hydroxysuccinimide activation,epoxide activation, sulfhydryl activation, hydrazide activation, andcarboxyl and amino derivatives for carbodiimide coupling chemistries.These and other solid media are well known and widely used in the art,and are available from commercial suppliers. Methods for bindingreceptor polypeptides to support media are well known in the art.Selection of a particular method is a matter of routine design and isdetermined in part by the properties of the chosen support. See, forexample, Affinity Chromatography: Principles & Methods, Pharmacia LKBBiotechnology, Uppsala, Sweden, 1988.

The polypeptides of the present invention can be isolated byexploitation of their physical properties. For example, immobilizedmetal ion adsorption (IMAC) chromatography can be used to purifyhistidine-rich proteins, including those comprising polyhistidine tags.Briefly, a gel is first charged with divalent metal ions to form achelate (Sulkowski, Trends in Biochem. 3:1-7, 1985). Histidine-richproteins will be adsorbed to this matrix with differing affinities,depending upon the metal ion used, and will be eluted by competitiveelution, lowering the pH, or use of strong chelating agents. Othermethods of purification include purification of glycosylated proteins bylectin affinity chromatography and ion exchange chromatography (Methodsin Enzymol., Vol. 182, “Guide to Protein Purification”, M. Deutscher,(ed.), Acad. Press, San Diego, 1990, pp. 529-39) and use of the solubleZCYTO18 receptor. Within additional embodiments of the invention, afusion of the polypeptide of interest and an affinity tag (e.g.,maltose-binding protein, an immunoglobulin domain) may be constructed tofacilitate purification.

Moreover, using methods described in the art, polypeptide fusions, orhybrid ZCYTO18 proteins, are constructed using regions or domains of theinventive ZCYTO18 in combination with those of other human cytokinefamily proteins (e.g. interleukins or GM-CSF), or heterologous proteins(Sambrook et al., ibid., Altschul et al., ibid., Picard, Cur. Opin.Biology, 5:511-5, 1994, and references therein). These methods allow thedetermination of the biological importance of larger domains or regionsin a polypeptide of interest. Such hybrids may alter reaction kinetics,binding, alter cell proliferative activity, constrict or expand thesubstrate specificity, or alter tissue and cellular localization of apolypeptide, and can be applied to polypeptides of unknown structure.

Fusion proteins can be prepared by methods known to those skilled in theart by preparing each component of the fusion protein and chemicallyconjugating them. Alternatively, a polynucleotide encoding bothcomponents of the fusion protein in the proper reading frame can begenerated using known techniques and expressed by the methods describedherein. For example, part or all of a helix conferring a biologicalfunction may be swapped between ZCYTO18 of the present invention withthe functionally equivalent helices from another family member, such asIL-10, zcyto10, MDA7, IL-15, IL-2, IL-4 and GM-CSF. Such componentsinclude, but are not limited to, the secretory signal sequence, helicesA, B, C, D and four-helical-bundle cytokines. Such fusion proteins wouldbe expected to have a biological functional profile that is the same orsimilar to polypeptides of the present invention or other knownfour-helical-bundle cytokine family proteins, depending on the fusionconstructed. Moreover, such fusion proteins may exhibit other propertiesas disclosed herein.

Standard molecular biological and cloning techniques can be used to swapthe equivalent domains between the ZCYTO18 polypeptide and thosepolypeptides to which they are fused. Generally, a DNA segment thatencodes a domain of interest, e.g., ZCYTO18 helices A through D, orother domain described herein, is operably linked in frame to at leastone other DNA segment encoding an additional polypeptide (for instance adomain or region from another cytokine, such as IL-10, or zcyto10, MDA7or the like), and inserted into an appropriate expression vector, asdescribed herein. Generally DNA constructs are made such that theseveral DNA segments that encode the corresponding regions of apolypeptide are operably linked in frame to make a single construct thatencodes the entire fusion protein, or a functional portion thereof. Forexample, a DNA construct would encode from N-terminus to C-terminus afusion protein comprising a signal polypeptide followed by a mature fourhelical bundle cytokine fusion protein containing helix A, followed byhelix B, followed by helix C, followed by helix D. or for example, anyof the above as interchanged with equivalent regions from another fourhelical bundle cytokine family protein. Such fusion proteins can beexpressed, isolated, and assayed for activity as described herein.Moreover, such fusion proteins can be used to express and secretefragments of the ZCYTO18 polypeptide, to be used, for example toinoculate an animal to generate anti-ZCYTO18 antibodies as describedherein. For example a secretory signal sequence can be operably linkedto helix A, B, C or D, or a combination thereof (e.g., operably linkedpolypeptides comprising helices A-B, B-C, C-D, A-C, A-D, B-D, or ZCYTO18polypeptide fragments described herein), to secrete a fragment ofZCYTO18 polypeptide that can be purified as described herein and serveas an antigen to be inoculated into an animal to produce anti-ZCYTO18antibodies, as described herein.

Zcyto18 polypeptides or fragments thereof may also be prepared throughchemical synthesis. ZCYTO18 polypeptides may be monomers or multimers;glycosylated or non-glycosylated; pegylated or non-pegylated; and may ormay not include an initial methionine amino acid residue. For example,the polypeptides can be prepared by solid phase peptide synthesis, forexample as described by Merrifield, J. Am. Chem. Soc. 85:2149, 1963.

The activity of molecules of the present invention can be measured usinga variety of assays that measure proliferation of and/or binding tocells expressing the ZCYTO18 receptor. Of particular interest arechanges in ZCYTO18-dependent cells. Suitable cell lines to be engineeredto be ZCYTO18-dependent include the IL-3-dependent BaF3 cell line(Palacios and Steinmetz, Cell 41: 727-734, 1985; Mathey-Prevot et al.,Mol. Cell. Biol. 6: 4133-4135, 1986), FDC-P1 (Hapel et al., Blood 64:786-790, 1984), and MO7e (Kiss et al., Leukemia 7: 235-240, 1993).Growth factor-dependent cell lines can be established according topublished methods (e.g. Greenberger et al., Leukemia Res. 8: 363-375,1984; Dexter et al., in Baum et al. Eds., Experimental Hematology Today,8th Ann. Mtg. Int. Soc. Exp. Hematol. 1979, 145-156, 1980). For example,Baf3 cells expressing the ZCYTO18 heterodimeric receptorzcytor11/CRF2-4, as described herein, can be used to assay the activityof ZCYTO18, ZCYTO18 receptor-binding fragments, and ZCYTO18 variants ofthe present invention. The BaF3 stable cell line that co-expressingzcytor11 and CRF2-4 (ZCYTO18 receptor) exhibits dose-dependentproliferative response to ZCYTO18 protein in the media without IL-3.

Proteins of the present invention are useful for stimulatingproliferation, activation, differentiation and/or induction orinhibition of specialized cell function of cells of the involvedhomeostasis of the hematopoiesis and immune function. In particular,ZCYTO18 polypeptides are useful for stimulating proliferation,activation, differentiation, induction or inhibition of specialized cellfunctions of cells of the hematopoetic lineages, including, but notlimited to, T cells, B cells, NK cells, dendritic cells, monocytes, andmacrophages. Proliferation and/or differentiation of hematopoietic cellscan be measured in vitro using cultured cells or in vivo byadministering molecules of the present invention to the appropriateanimal model. Assays measuring cell proliferation or differentiation arewell known in the art. For example, assays measuring proliferationinclude such assays as chemosensitivity to neutral red dye (Cavanaugh etal., Investigational New Drugs 8:347-354, 1990, incorporated herein byreference), incorporation of radiolabelled nucleotides (Cook et al.,Analytical Biochem. 179:1-7, 1989, incorporated herein by reference),incorporation of 5-bromo-2′-deoxyuridine (BrdU) in the DNA ofproliferating cells (Porstmann et al., J. Immunol. Methods 82:169-179,1985, incorporated herein by reference), and use of tetrazolium salts(Mosmann, J. Immunol. Methods 65:55-63, 1983; Alley et al., Cancer Res.48:589-601, 1988; Marshall et al., Growth Reg. 5:69-84, 1995; andScudiero et al., Cancer Res. 48:4827-4833, 1988; all incorporated hereinby reference). Assays measuring differentiation include, for example,measuring cell-surface markers associated with stage-specific expressionof a tissue, enzymatic activity, functional activity or morphologicalchanges (Watt, FASEB, 5:281-284, 1991; Francis, Differentiation57:63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses,161-171, 1989; all incorporated herein by reference).

IL-10 is a cytokine that inhibits production of other cytokines, inducesproliferation and differentiation of activated B lymphocytes, inhibitsHIV-1 replication and exhibits antagonistic effects on gamma interferon.IL-10 appears to exist as a dimer formed from two alpha-helicalpolypeptide regions related by a 180° rotation. See, for example, Zdanovet al., Structure: 3(6): 591-601 (1996). IL-10 has been reported to be aproduct of activated Th2 T-cells, B-cells, keratinocytes andmonocytes/macrophages that is capable of modulating a Th1 T-cellresponse. Such modulation may be accomplished by inhibiting cytokinesynthesis by Th1 T-cells. See, for example, Hus et al., Int. Immunol. 4:563 (1992) and D'Andrea et al., J. Exp. Med. 178: 1042 (1992). IL-10 hasalso been reported to inhibit cytokine synthesis by natural killer cellsand monocytes/macrophages. See, for example, Hus et al. cited above andFiorentino et al., J. Immunol. 146: 3444 (1991). In addition, IL-10 hasbeen found to have a protective effect with respect to insulin dependentdiabetes mellitus. Similarly, as a cytokine sharing polypeptidestructure and some sequence similarity to IL-10, ZCYTO18 can have theseabove disclosed activities, and the assays used to assess IL-10 activitycan be applied to assay ZCYTO18 activity.

The molecules of the present invention can be assayed in vivo usingviral delivery systems. Exemplary viruses for this purpose includeadenovirus, herpesvirus, retroviruses, vaccinia virus, andadeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus,is currently the best studied gene transfer vector for delivery ofheterologous nucleic acid (for review, see T. C. Becker et al., Meth.Cell Biol. 43:161-89, 1994; and J. T. Douglas and D. T. Curiel, Science& Medicine 4:44-53, 1997). The adenovirus system offers severaladvantages: (i) adenovirus can accommodate relatively large DNA inserts;(ii) can be grown to high-titer; (iii) infect a broad range of mammaliancell types; and (iv) can be used with many different promoters includingubiquitous, tissue specific, and regulatable promoters. Also, becauseadenoviruses are stable in the bloodstream, they can be administered byintravenous injection.

Using adenovirus vectors where portions of the adenovirus genome aredeleted, inserts are incorporated into the viral DNA by direct ligationor by homologous recombination with a co-transfected plasmid. In anexemplary system, the essential E1 gene has been deleted from the viralvector, and the virus will not replicate unless the E1 gene is providedby the host cell (the human 293 cell line is exemplary). Whenintravenously administered to intact animals, adenovirus primarilytargets the liver. If the adenoviral delivery system has an E1 genedeletion, the virus cannot replicate in the host cells. However, thehost's tissue (e.g., liver) will express and process (and, if asecretory signal sequence is present, secrete) the heterologous protein.Secreted proteins will enter the circulation in the highly vascularizedliver, and effects on the infected animal can be determined.

Moreover, adenoviral vectors containing various deletions of viral genescan be used in an attempt to reduce or eliminate immune responses to thevector. Such adenoviruses are E1 deleted, and in addition containdeletions of E2A or E4 (Lusky, M. et al., J. Virol. 72:2022-2032, 1998;Raper, S. E. et al., Human Gene Therapy 9:671-679, 1998). In addition,deletion of E2b is reported to reduce immune responses (Amalfitano, A.et al., J. Virol. 72:926-933, 1998). Moreover, by deleting the entireadenovirus genome, very large inserts of heterologous DNA can beaccommodated. Generation of so called “gutless” adenoviruses where allviral genes are deleted are particularly advantageous for insertion oflarge inserts of heterologous DNA. For review, see Yeh, P. andPerricaudet, M., FASEB J. 11:615-623, 1997.

The adenovirus system can also be used for protein production in vitro.By culturing adenovirus-infected cells under conditions where the cellsare not rapidly dividing, the cells can produce proteins for extendedperiods of time. For instance, BHK cells are grown to confluence in cellfactories, then exposed to the adenoviral vector encoding the secretedprotein of interest. The cells are then grown under serum-freeconditions, which allows infected cells to survive for several weekswithout significant cell division. Alternatively, adenovirus vectorinfected 293 cells can be grown as adherent cells or in suspensionculture at relatively high cell density to produce significant amountsof protein (See Garnier et al., Cytotechnol. 15:145-55, 1994). Witheither protocol, an expressed, secreted heterologous protein can berepeatedly isolated from the cell culture supernatant, lysate, ormembrane fractions depending on the disposition of the expressed proteinin the cell. Within the infected 293 cell production protocol,non-secreted proteins may also be effectively obtained.

In view of the tissue distribution observed for ZCYTO18 receptoragonists (including the natural ligand/substrate/cofactor/etc.) and/orantagonists have enormous potential in both in vitro and in vivoapplications. Compounds identified as ZCYTO18 agonists are useful forexpansion, proliferation, activation, differentiation, and/or inductionor inhibition of specialized cell functions of cells involved inhomeostasis of hematopoiesis and immune function. For example, ZCYTO18and agonist compounds are useful as components of defined cell culturemedia, and may be used alone or in combination with other cytokines andhormones to replace serum that is commonly used in cell culture.Agonists are thus useful in specifically promoting the growth and/ordevelopment of T-cells, B-cells, and other cells of the lymphoid andmyeloid lineages in culture.

Antagonists are also useful as research reagents for characterizingsites of ligand-receptor interaction. Antagonists are useful to inhibitexpansion, proliferation, activation, and/or differentiation of cellsinvolved in regulating hematopoiesis. Inhibitors of ZCYTO18 activity(ZCYTO18 antagonists) include anti-ZCYTO18 antibodies and solubleZCYTO18 receptors, as well as other peptidic and non-peptidic agents(including ribozymes).

ZCYTO18 can also be used to identify inhibitors (antagonists) of itsactivity. Test compounds are added to the assays disclosed herein toidentify compounds that inhibit the activity of ZCYTO18. In addition tothose assays disclosed herein, samples can be tested for inhibition ofZCYTO18 activity within a variety of assays designed to measure receptorbinding, the stimulation/inhibition of ZCYTO18-dependent cellularresponses or proliferation of ZCYTO18 receptor-expressing cells.

A ZCYTO18 polypeptide can be expressed as a fusion with animmunoglobulin heavy chain constant region, typically an F_(C) fragment,which contains two constant region domains and lacks the variableregion. Methods for preparing such fusions are disclosed in U.S. Pat.Nos. 5,155,027 and 5,567,584. Such fusions are typically secreted asmultimeric molecules wherein the Fc portions are disulfide bonded toeach other and two non-Ig polypeptides are arrayed in closed proximityto each other. Fusions of this type can be used to (e.g., fordimerization, increasing stability and in vivo half-life, affinitypurify ligand, in vitro assay tool, antagonist). For use in assays, thechimeras are bound to a support via the F_(C) region and used in anELISA format. Fc fusions may represent preferred therapeutic proteinswith different pharmacokinetics and altered action.

Polypeptides containing the receptor-binding region of the ligand can beused for purification of receptor. The ligand polypeptide is immobilizedon a solid support, such as beads of agarose, cross-linked agarose,glass, cellulosic resins, silica-based resins, polystyrene, cross-linkedpolyacrylamide, or like materials that are stable under the conditionsof use. Methods for linking polypeptides to solid supports are known inthe art, and include amine chemistry, cyanogen bromide activation,N-hydroxysuccinimide activation, epoxide activation, sulfhydrylactivation, and hydrazide activation. The resulting media will generallybe configured in the form of a column, and fluids containing receptorsare passed through the column one or more times to allow receptor tobind to the ligand polypeptide. The receptor is then eluted usingchanges in salt concentration, chaotropic agents (MnCl₂), or pH todisrupt ligand-receptor binding.

ZCYTO18 polypeptides or ZCYTO18 fusion proteins are used, for example,to identify the ZCYTO18 receptor. Using labeled ZCYTO18 polypeptides,cells expressing the receptor are identified by fluorescenceimmunocytometry or immunohistochemistry. ZCYTO18 polypeptides are usefulin determining the distribution of the receptor on tissues or specificcell lineages, and to provide insight into receptor/ligand biology. Anexemplary method to identify a ZCYTO18 receptor in vivo or in vitro,e.g., in cell lines, is to us a ZCYTO18 polypeptide fused to thecatalytic domain of Alkaline phosphatase (AP), as described in Feiner,L. et al., Neuron 19:539-545, 1997. Such AP fusions, as well asradiolabeled ZCYTO18, ZCYTO18 fusions with fluorescent lables, andothers described herein, combined with standard cloning techniquesenable one of skill in the art to visualize, identify and clone theZCYTO18 receptor.

Conversely, a ZCYTO18-binding polypeptide can be used for purificationof ligand. The polypeptide is immobilized on a solid support, such asbeads of agarose, cross-linked agarose, glass, cellulosic resins,silica-based resins, polystyrene, cross-linked polyacrylamide, or likematerials that are stable under the conditions of use. Methods forlinking polypeptides to solid supports are known in the art, and includeamine chemistry, cyanogen bromide activation, N-hydroxysuccinimideactivation, epoxide activation, sulfhydryl activation, and hydrazideactivation. The resulting medium will generally be configured in theform of a column, and fluids containing ligand are passed through thecolumn one or more times to allow ligand to bind to the receptorpolypeptide. The ligand is then eluted using changes in saltconcentration, chaotropic agents (guanidine HCl), or pH to disruptligand-receptor binding.

An assay system that uses a ligand-binding receptor (or an antibody, onemember of a complement/anti-complement pair) or a binding fragmentthereof, and a commercially available biosensor instrument (BIAcore,Pharmacia Biosensor, Piscataway, N.J.) may be advantageously employed.Such receptor, antibody, member of a complement/anti-complement pair orfragment is immobilized onto the surface of a receptor chip. Use of thisinstrument is disclosed by Karlsson, J. Immunol. Methods 145:229-40,1991 and Cunningham and Wells, J. Mol. Biol. 234:554-63, 1993. Areceptor, antibody, member or fragment is covalently attached, usingamine or sulfhydryl chemistry, to dextran fibers that are attached togold film within the flow cell. A test sample is passed through thecell. If a ligand, epitope, or opposite member of thecomplement/anti-complement pair is present in the sample, it will bindto the immobilized receptor, antibody or member, respectively, causing achange in the refractive index of the medium, which is detected as achange in surface plasmon resonance of the gold film. This system allowsthe determination of on- and off-rates, from which binding affinity canbe calculated, and assessment of stoichiometry of binding.Alternatively, ligand/receptor binding can be analyzed using SELDI™technology (Ciphergen, Inc., Palo Alto, Calif.).

Ligand-binding receptor polypeptides can also be used within other assaysystems known in the art. Such systems include Scatchard analysis fordetermination of binding affinity (see Scatchard, Ann. NY Acad. Sci. 51:660-72, 1949) and calorimetric assays (Cunningham et al., Science253:545-48, 1991; Cunningham et al., Science 245:821-25, 1991).

Zcyto18 polypeptides can also be used to prepare antibodies that bind toZCYTO18 epitopes, peptides or polypeptides. The ZCYTO18 polypeptide or afragment thereof serves as an antigen (immunogen) to inoculate an animaland elicit an immune response. One of skill in the art would recognizethat antigenic, epitope-bearing polypeptides contain a sequence of atleast 6, preferably at least 9, and more preferably at least 15 to about30 contiguous amino acid residues of a ZCYTO18 polypeptide (e.g., SEQ IDNO:3). Polypeptides comprising a larger portion of a ZCYTO18polypeptide, i.e., from 30 to 100 residues up to the entire length ofthe amino acid sequence are included. Antigens or immunogenic epitopescan also include attached tags, adjuvants and carriers, as describedherein. Suitable antigens include the ZCYTO18 polypeptide encoded by SEQID NO:3 from amino acid number 23 to amino acid number 167, or acontiguous 9 to 144, or 30 to 144 amino acid fragment thereof. Othersuitable antigens include helices of the four-helical-bundle structure,as described herein. Preferred peptides to use as antigens arehydrophilic peptides such as those predicted by one of skill in the artfrom a hydrophobicity plot, as described herein. For example suitablehydrophilic peptides include: (1) amino acid number 29 (Arg) to aminoacid number 34 (Asn) of SEQ ID NO:3; (2) amino acid number 121 (His) toamino acid number 126 (Asp) of SEQ ID NO:3; (3) amino acid number 134(Gln) to amino acid number 139 (Thr) of SEQ ID NO:3; (4) amino acidnumber 137 (Lys) to amino acid number 142 (Lys) of SEQ ID NO:3; and (5)amino acid number 145 (Glu) to amino acid number 150 (Lys) of SEQ IDNO:2. Moreover, ZCYTO18 antigenic epitopes as predicted by aJameson-Wolf plot, e.g., using DNASTAR Protean program (DNASTAR, Inc.,Madison, Wis.) serve as preferred antigens, and are readily determinedby one of skill in the art.

Antibodies from an immune response generated by inoculation of an animalwith these antigens (or immunogens) can be isolated and purified asdescribed herein. Methods for preparing and isolating polyclonal andmonoclonal antibodies are well known in the art. See, for example,Current Protocols in Immunology, Cooligan, et al. (eds.), NationalInstitutes of Health, John Wiley and Sons, Inc., 1995; Sambrook et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., 1989; and Hurrell, J. G. R., Ed., Monoclonal HybridomaAntibodies: Techniques and Applications, CRC Press, Inc., Boca Raton,Fla., 1982.

As would be evident to one of ordinary skill in the art, polyclonalantibodies can be generated from inoculating a variety of warm-bloodedanimals such as horses, cows, goats, sheep, dogs, chickens, rabbits,mice, and rats with a ZCYTO18 polypeptide or a fragment thereof. Theimmunogenicity of a ZCYTO18 polypeptide may be increased through the useof an adjuvant, such as alum (aluminum hydroxide) or Freund's completeor incomplete adjuvant. Polypeptides useful for immunization alsoinclude fusion polypeptides, such as fusions of ZCYTO18 or a portionthereof with an immunoglobulin polypeptide or with maltose bindingprotein. The polypeptide immunogen may be a full-length molecule or aportion thereof. If the polypeptide portion is “hapten-like”, suchportion may be advantageously joined or linked to a macromolecularcarrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin(BSA) or tetanus toxoid) for immunization.

As used herein, the term “antibodies” includes polyclonal antibodies,affinity-purified polyclonal antibodies, monoclonal antibodies, andantigen-binding fragments, such as F(ab′)₂ and Fab proteolyticfragments. Genetically engineered intact antibodies or fragments, suchas chimeric antibodies, Fv fragments, single chain antibodies and thelike, as well as synthetic antigen-binding peptides and polypeptides,are also included. Non-human antibodies may be humanized by graftingnon-human CDRs onto human framework and constant regions, or byincorporating the entire non-human variable domains (optionally“cloaking” them with a human-like surface by replacement of exposedresidues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced. Moreover, human antibodies can beproduced in transgenic, non-human animals that have been engineered tocontain human immunoglobulin genes as disclosed in WIPO Publication WO98/24893. It is preferred that the endogenous immunoglobulin genes inthese animals be inactivated or eliminated, such as by homologousrecombination.

Antibodies are considered to be specifically binding if: 1) they exhibita threshold level of binding activity, and 2) they do not significantlycross-react with related polypeptide molecules. A threshold level ofbinding is determined if anti-ZCYTO18 antibodies herein bind to aZCYTO18 polypeptide, peptide or epitope with an affinity at least10-fold greater than the binding affinity to control (non-ZCYTO18)polypeptide. It is preferred that the antibodies exhibit a bindingaffinity (K_(a)) of 10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ or greater,more preferably 10⁸ M⁻¹ or greater, and most preferably 10⁹ M⁻¹ orgreater. The binding affinity of an antibody can be readily determinedby one of ordinary skill in the art, for example, by Scatchard analysis(Scatchard, G., Ann. NY Acad. Sci. 51: 660-672, 1949).

Whether anti-ZCYTO18 antibodies do not significantly cross-react withrelated polypeptide molecules is shown, for example, by the antibodydetecting ZCYTO18 polypeptide but not known related polypeptides using astandard Western blot analysis (Ausubel et al., ibid.). Examples ofknown related polypeptides are those disclosed in the prior art, such asknown orthologs, and paralogs, and similar known members of a proteinfamily. Screening can also be done using non-human ZCYTO18, and ZCYTO18mutant polypeptides. Moreover, antibodies can be “screened against”known related polypeptides, to isolate a population that specificallybinds to the ZCYTO18 polypeptides. For example, antibodies raised toZCYTO18 are adsorbed to related polypeptides adhered to insolublematrix; antibodies specific to ZCYTO18 will flow through the matrixunder the proper buffer conditions. Screening allows isolation ofpolyclonal and monoclonal antibodies non-crossreactive to known closelyrelated polypeptides (Antibodies: A Laboratory Manual, Harlow and Lane(eds.), Cold Spring Harbor Laboratory Press, 1988; Current Protocols inImmunology, Cooligan, et al. (eds.), National Institutes of Health, JohnWiley and Sons, Inc., 1995). Screening and isolation of specificantibodies is well known in the art. See, Fundamental Immunology, Paul(eds.), Raven Press, 1993; Getzoff et al., Adv. in Immunol. 43: 1-98,1988; Monoclonal Antibodies: Principles and Practice, Goding, J. W.(eds.), Academic Press Ltd., 1996; Benjamin et al., Ann. Rev. Immunol2^(.) 67-101, 1984. Specifically binding anti-ZCYTO18 antibodies can bedetected by a number of methods in the art, and disclosed below.

A variety of assays known to those skilled in the art can be utilized todetect antibodies which bind to ZCYTO18 proteins or polypeptides.Exemplary assays are described in detail in Antibodies: A LaboratoryManual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press,1988. Representative examples of such assays include: concurrentimmunoelectrophoresis, radioimmunoassay, radioimmuno-precipitation,enzyme-linked immunosorbent assay (ELISA), dot blot or Western blotassay, inhibition or competition assay, and sandwich assay. In addition,antibodies can be screened for binding to wild-type versus mutantZCYTO18 protein or polypeptide.

Alternative techniques for generating or selecting antibodies usefulherein include in vitro exposure of lymphocytes to ZCYTO18 protein orpeptide, and selection of antibody display libraries in phage or similarvectors (for instance, through use of immobilized or labeled ZCYTO18protein or peptide). Genes encoding polypeptides having potentialZCYTO18 polypeptide binding domains can be obtained by screening randompeptide libraries displayed on phage (phage display) or on bacteria,such as E. coli. Nucleotide sequences encoding the polypeptides can beobtained in a number of ways, such as through random mutagenesis andrandom polynucleotide synthesis. These random peptide display librariescan be used to screen for peptides which interact with a known targetwhich can be a protein or polypeptide, such as a ligand or receptor, abiological or synthetic macromolecule, or organic or inorganicsubstances. Techniques for creating and screening such random peptidedisplay libraries are known in the art (Ladner et al., U.S. Pat. No.5,223,409; Ladner et al., U.S. Pat. No. 4,946,778; Ladner et al., U.S.Pat. No. 5,403,484 and Ladner et al., U.S. Pat. No. 5,571,698) andrandom peptide display libraries and kits for screening such librariesare available commercially, for instance from Clontech (Palo Alto,Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc.(Beverly, Mass.) and Pharmacia LKB Biotechnology Inc. (Piscataway,N.J.). Random peptide display libraries can be screened using theZCYTO18 sequences disclosed herein to identify proteins which bind toZCYTO18. These “binding polypeptides” which interact with ZCYTO18polypeptides can be used for tagging cells; for isolating homologpolypeptides by affinity purification; they can be directly orindirectly conjugated to drugs, toxins, radionuclides and the like.These binding polypeptides can also be used in analytical methods suchas for screening expression libraries and neutralizing activity, e.g.,for blocking interaction between ligand and receptor, or viral bindingto a receptor. The binding polypeptides can also be used for diagnosticassays for determining circulating levels of ZCYTO18 polypeptides; fordetecting or quantitating soluble ZCYTO18 polypeptides as marker ofunderlying pathology or disease. These binding polypeptides can also actas ZCYTO18 “antagonists” to block ZCYTO18 binding and signaltransduction in vitro and in vivo. These anti-ZCYTO18 bindingpolypeptides would be useful for inhibiting ZCYTO18 activity orprotein-binding.

Antibodies to ZCYTO18 may be used for tagging cells that expressZCYTO18; for isolating ZCYTO18 by affinity purification; for diagnosticassays for determining circulating levels of ZCYTO18 polypeptides; fordetecting or quantitating soluble ZCYTO18 as a marker of underlyingpathology or disease; in analytical methods employing FACS; forscreening expression libraries; for generating anti-idiotypicantibodies; and as neutralizing antibodies or as antagonists to blockZCYTO18 activity in vitro and in vivo. Suitable direct tags or labelsinclude radionuclides, enzymes, substrates, cofactors, inhibitors,fluorescent markers, chemiluminescent markers, magnetic particles andthe like; indirect tags or labels may feature use of biotin-avidin orother complement/anti-complement pairs as intermediates. Antibodiesherein may also be directly or indirectly conjugated to drugs, toxins,radionuclides and the like, and these conjugates used for in vivodiagnostic or therapeutic applications. Moreover, antibodies to ZCYTO18or fragments thereof may be used in vitro to detect denatured ZCYTO18 orfragments thereof in assays, for example, Western Blots or other assaysknown in the art.

Antibodies or polypeptides herein can also be directly or indirectlyconjugated to drugs, toxins, radionuclides and the like, and theseconjugates used for in vivo diagnostic or therapeutic applications. Forinstance, polypeptides or antibodies of the present invention can beused to identify or treat tissues or organs that express a correspondinganti-complementary molecule (receptor or antigen, respectively, forinstance). More specifically, ZCYTO18 polypeptides or anti-ZCYTO18antibodies, or bioactive fragments or portions thereof, can be coupledto detectable or cytotoxic molecules and delivered to a mammal havingcells, tissues or organs that express the anti-complementary molecule.

Suitable detectable molecules may be directly or indirectly attached tothe polypeptide or antibody, and include radionuclides, enzymes,substrates, cofactors, inhibitors, fluorescent markers, chemiluminescentmarkers, magnetic particles and the like. Suitable cytotoxic moleculesmay be directly or indirectly attached to the polypeptide or antibody,and include bacterial or plant toxins (for instance, diphtheria toxin,Pseudomonas exotoxin, ricin, abrin and the like), as well as therapeuticradionuclides, such as iodine-131, rhenium-188 or yttrium-90 (eitherdirectly attached to the polypeptide or antibody, or indirectly attachedthrough means of a chelating moiety, for instance). Polypeptides orantibodies may also be conjugated to cytotoxic drugs, such asadriamycin. For indirect attachment of a detectable or cytotoxicmolecule, the detectable or cytotoxic molecule can be conjugated with amember of a complementary/anticomplementary pair, where the other memberis bound to the polypeptide or antibody portion. For these purposes,biotin/streptavidin is an exemplary complementary/anticomplementarypair.

In another embodiment, polypeptide-toxin fusion proteins orantibody-toxin fusion proteins can be used for targeted cell or tissueinhibition or ablation (for instance, to treat cancer cells or tissues).Alternatively, if the polypeptide has multiple functional domains (i.e.,an activation domain or a receptor binding domain, plus a targetingdomain), a fusion protein including only the targeting domain may besuitable for directing a detectable molecule, a cytotoxic molecule or acomplementary molecule to a cell or tissue type of interest. Ininstances where the domain only fusion protein includes a complementarymolecule, the anti-complementary molecule can be conjugated to adetectable or cytotoxic molecule. Such domain-complementary moleculefusion proteins thus represent a generic targeting vehicle forcell/tissue-specific delivery of genericanti-complementary-detectable/cytotoxic molecule conjugates. Suchcytokine toxin fusion proteins can be used for in vivo killing of targettissues.

In another embodiment, ZCYTO18 cytokine fusion proteins orantibody-cytokine fusion proteins can be used for in vivo killing oftarget tissues (for example, leukemia, lymphoma, lung cancer, coloncancer, melanoma, pancreatic cancer, ovanian cancer, blood and bonemarrow cancers, or other cancers wherein ZCYTO18 receptors ar expressed)(See, generally, Hornick et al., Blood 89:4437-47, 1997). The describedfusion proteins enable targeting of a cytokine to a desired site ofaction, thereby providing an elevated local concentration of cytokine.Suitable ZCYTO18 polypeptides or anti-ZCYTO18 antibodies target anundesirable cell or tissue (i.e., a tumor or a leukemia), and the fusedcytokine mediated improved target cell lysis by effector cells. Suitablecytokines for this purpose include interleukin 2 andgranulocyte-macrophage colony-stimulating factor (GM-CSF), for instance.

In yet another embodiment, if the ZCYTO18 polypeptide or anti-ZCYTO18antibody targets vascular cells or tissues, such polypeptide or antibodymay be conjugated with a radionuclide, and particularly with abeta-emitting radionuclide, to reduce restenosis. Such therapeuticapproaches pose less danger to clinicians who administer the radioactivetherapy. For instance, iridium-192 impregnated ribbons placed intostented vessels of patients until the required radiation dose wasdelivered showed decreased tissue growth in the vessel and greaterluminal diameter than the control group, which received placebo ribbons.Further, revascularisation and stent thrombosis were significantly lowerin the treatment group. Similar results are predicted with targeting ofa bioactive conjugate containing a radionuclide, as described herein.

The bioactive polypeptide or antibody conjugates described herein can bedelivered intravenously, intraarterially or intraductally, or may beintroduced locally at the intended site of action.

Zcyto18 was isolated from tissue known to have important immunologicalfunction and which contain cells which play a role in the immune system.ZCYTO18 ligand is expressed in CD3+ selected, activated peripheral bloodcells. This suggests that ZCYTO18 expression may be regulated andincrease after T cell activation. Moreover, polypeptides of the presentinvention may have an effect on the growth/expansion and/ordifferentiated state of T- or B-Cells, T- or B-cell progenitors, NKcells or NK progenitors. Moreover, ZCYTO18 can effect proliferationand/or differentiation of T cells and B cells in vivo. Factor that bothstimulate proliferation of hematopoietic progenitors and activate maturecells are generally known. NK cells are responsive to IL-2 alone, butproliferation and activation generally require additional growthfactors. For example, it has been shown that IL-7 and Steel Factor(c-kit ligand) were required for colony formation of NK progenitors.IL-15+IL-2 in combination with IL-7 and Steel Factor was more effective(Mrózek et al., Blood 87:2632-2640, 1996). However, unidentifiedcytokines may be necessary for proliferation of specific subsets of NKcells and/or NK progenitors (Robertson et. al., Blood 76:2451-2438,1990). A composition comprising ZCYTO18 and IL-15 may stimulate NKprogenitors and NK cells, as a composition that is more potent thanpreviously described factors and combinations of factors. Similarly,such combinations of factors that include ZCYTO18 may also affect otherhematopoietic and lymphoid cell types, such as T-cells, B-cells,macrophages, dendritic cells, and the like.

Most four-helix bundle cytokines as well as other proteins produced byactivated lymphocytes play an important biological role in celldifferentiation, activation, recruitment and homeostasis of cellsthroughout the body. Therapeutic utility includes treatment of diseaseswhich require immune regulation including autoimmune diseases, such as,rheumatoid arthritis, multiple sclerosis, myasthenia gravis, systemiclupus erythomatosis (SLE) and diabetes. Zcyto18 may be important in theregulation of inflammation, and therefore would be useful in treatingrheumatoid arthritis, asthma, ulcerative colitis, inflammatory boweldisease, Crohn's disease, pancreatitis, and sepsis. There may be a roleof ZCYTO18 in mediating tumor cell killing and therefore would be usefulin the treatment of cancer such as ovarian cancer, lung cancer,melanoma, and colon cancer. Zcyto18 may be a potential therapeutic insuppressing the immune system which would be important for reducinggraft rejection. Zcyto18 may have usefulness in prevention ofgraft-vs-host disease.

The proteins of the present invention can also be used ex vivo, such asin autologous marrow culture. Briefly, bone marrow is removed from apatient prior to chemotherapy or organ transplant and treated withZCYTO18, optionally in combination with one or more other cytokines. Thetreated marrow is then returned to the patient after chemotherapy tospeed the recovery of the marrow or after transplant to suppress graftvs. Host disease. In addition, the proteins of the present invention canalso be used for the ex vivo expansion of marrow or peripheral bloodprogenitor (PBPC) cells. Prior to treatment, marrow can be stimulatedwith stem cell factor (SCF) to release early progenitor cells intoperipheral circulation. These progenitors can be collected andconcentrated from peripheral blood and then treated in culture withZCYTO18, optionally in combination with one or more other cytokines,including but not limited to IL-10, zcyto10, MDA7, SCF, IL-2, IL-4, IL-7or IL-15, to differentiate and proliferate into high-density lymphoidcultures, which can then be returned to the patient followingchemotherapy or transplantation.

Alternatively, ZCYTO18 may activate the immune system which would beimportant in boosting immunity to infectious diseases, treatingimmunocompromised patients, such as HIV+patients, or in improvingvaccines. In particular, ZCYTO18 stimulation or expansion of T-cells,B-cells, NK cells, and the like, or their progenitors, would providetherapeutic value in treatment of viral infection, and as ananti-neoplastic factor. NK cells are thought to play a major role inelimination of metastatic tumor cells and patients with both metastasesand solid tumors have decreased levels of NK cell activity (Whitesideet. al., Curr. Top. Microbiol. Immunol 230:221-244, 1998).

Further analysis of mice injected with ZCYTO18 adenovirus reveals thatalbumin levels are reduced relative to control adenovirus injectedanimals, and glucose levels are depressed significantly. However liverenzymes (ALT and AST) are at similar levels to those seen for miceinjected with control adenovirus. ZCYTO18 may specifically inhibit oralter liver cell functions. Alternatively excess ZCYTO18 may synergizewith viral infection leading to adverse effects on the liver. Thusantagonists (antibodies, muteins, soluble receptors) may be useful totreat viral disease. Especially viral diseases that target the liversuch as: Hepatitis B, Hepatitis C and Adenovirus. Viral disease in othertissues may be treated with antagonists to ZCYTO18, for example viralmeningitis, and HIV-related disease.

Mice injected with ZCYTO18 adenovirus display weight-loss, loss ofmobility and a failure to groom, and a reduction in circulatinglymphocytes. These changes are typical of those seen during septic shockand other inflammatory conditions. These effects may be caused directlyby ZCYTO18 or indirectly by induction of elevated levels ofproinflammatory cytokines such as IL-1, TNFα, and IL-6. Antagonists toZCYTO18 may be useful to treat septic shock, adult respiratory distresssyndrome, endotoxemia, and meningitis. Other diseases that may benefitfrom ZCYTO18 antagonists include: Hemorrhagic shock, disseminatedintravascular coagulopathy, myocardial ischemia, stroke, rejection oftransplanted organs, pulmonary fibrosis, inflammatory hyperalgesia andcachexia.

Mice injected with ZCYTO18 adenovirus display reduced numbers ofperipheral blood lymphocytes. This is likely to be a direct inhibitoryeffect of ZCYTO18 on peripheral blood lymphocytes. Antagonizing ZCYTO18may promote lymphocyte maintenance and growth especially when they areneeded to eradicate bacterial, viral or parasitic pathogens. Thusantagonizing ZCYTO18 may benefit patients with: Tuberculosis,cryptogenic fibrosing alveolitis, pneumonia, meningococal disease, AIDS,HIV-related lung disease, hepatitis, viral meningitis, malaria, anddysentery (Shigella dysenteriae).

The lymphocyte inhibitory effects of ZCYTO18 may be used to reduceautoimmunity and to inhibit the growth of lymphoma tumors, especiallynon-Hodgkin's lymphoma and lymphoid leukemias. ZCYTO18 may also be usedto inhibit lymphocytes and promote graft acceptance for organ transplantpatients. Kidney and bone marrow grafts would be suitable indications.

Mice injected with ZCYTO18 adenovirus display significantly increasednumbers of platelets. Mild bleeding disorders (MBDs) associated withplatelet dysfunctions are relatively common (Bachmann, Seminars inHematology 17: 292-305, 1980), as are a number of congenital disordersof platelet function, including Bernard-Soulier syndrome (deficiency inplatelet GPIb), Glanzmann's thrombasthenia (deficiency of GPIIb andGPIIIa), congenital afibrinogenemia (diminished or absent levels offibrinogen in plasma and platelets), and gray platelet syndrome (absenceof a-granules). In addition there are a number of disorders associatedwith platelet secretion, storage pool deficiency, abnormalities inplatelet arachidonic acid pathway, deficiencies of plateletcyclooxygenase and thromboxane synthetase and defects in plateletactivation (reviewed by Rao and Holmsen, Seminars in Hematology 23:102-118, 1986).

The proteins of the present invention were shown to increase plateletand neutrophils in vivo in animals, and can be used therapeuticallywherever it is desirable to increase the level of platelets andneutrophils, such as in the treatment of cytopenia, such as that inducedby aplastic anemia, myelodisplastic syndromes, chemotherapy orcongenital cytopenias. The proteins are also useful for increasingplatelet production, such as in the treatment of thrombocytopenia.Thrombocytopenia is associated with a diverse group of diseases andclinical situations that may act alone or in concert to produce thecondition. Lowered platelet counts can result from, for example, defectsin platelet production, abnormal platelet distribution, dilutionallosses due to massive transfusions, or abnormal destruction ofplatelets. For example, chemotherapeutic drugs used in cancer therapymay suppress development of platelet progenitor cells in the bonemarrow, and the resulting thrombocytopenia limits the chemotherapy andmay necessitate transfusions. In addition, certain malignancies canimpair platelet production and platelet distribution. Radiation therapyused to kill malignant cells also kills platelet progenitor cells.Thrombocytopenia may also arise from various platelet autoimmunedisorders induced by drugs, neonatal alloimmunity or platelettransfusion alloimmunity. The proteins of the present invention canreduce or eliminate the need for transfusions, thereby reducing theincidence of platelet alloimmunity. Abnormal destruction of plateletscan result from: (1) increased platelet consumption in vascular graftsor traumatized tissue; or (2) immune mechanisms associated with, forexample, drug-induced thrombocytopenia, idiopathic thrombocytopenicpurpura (ITP), autoimmune diseases, hematologic disorders such asleukemia and lymphoma or metastatic cancers involving bone marrow. Otherindications for the proteins of the present invention include aplasticanemia and drug-induced marrow suppression resulting from, for example,chemotherapy or treatment of HIV infection with AZT.

Thrombocytopenia is manifested as increased bleeding, such as mucosalbleedings from the nasal-oral area or the gastrointestinal tract, aswell as oozing from wounds, ulcers or injection sites.

For pharmaceutical use, the proteins of the present invention areformulated for parenteral, particularly intravenous or subcutaneous,delivery according to conventional methods. Intravenous administrationwill be by bolus injection, controlled release, e.g, using mini-pumps orother appropriate technology, or by infusion over a typical period ofone to several hours. In general, pharmaceutical formulations willinclude a hematopoietic protein in combination with a pharmaceuticallyacceptable vehicle, such as saline, buffered saline, 5% dextrose inwater or the like. Formulations may further include one or moreexcipients, preservatives, solubilizers, buffering agents, albumin toprevent protein loss on vial surfaces, etc. In addition, thehematopoietic proteins of the present invention may be combined withother cytokines, particularly early-acting cytokines such as stem cellfactor, IL-3, IL-6, IL-11 or GM-CSF. When utilizing such a combinationtherapy, the cytokines may be combined in a single formulation or may beadministered in separate formulations. Methods of formulation are wellknown in the art and are disclosed, for example, in Remington'sPharmaceutical Sciences, Gennaro, ed., Mack Publishing Co., Easton Pa.,1990, which is incorporated herein by reference. Therapeutic doses willgenerally be in the range of 0.1 to 100 mg/kg of patient weight per day,preferably 0.5-20 mg/kg per day, with the exact dose determined by theclinician according to accepted standards, taking into account thenature and severity of the condition to be treated, patient traits, etc.Determination of dose is within the level of ordinary skill in the art.The proteins will commonly be administered over a period of up to 28days following chemotherapy or bone-marrow transplant or until aplatelet count of >20,000/mm³, preferably >50,000/mm³, is achieved. Morecommonly, the proteins will be administered over one week or less, oftenover a period of one to three days. In general, a therapeuticallyeffective amount of ZCYTO18 is an amount sufficient to produce aclinically significant increase in the proliferation and/ordifferentiation of lymphoid or myeloid progenitor cells, which will bemanifested as an increase in circulating levels of mature cells (e.g.platelets or neutrophils). Treatment of platelet disorders will thus becontinued until a platelet count of at least 20,000/mm³, preferably50,000/mm³, is reached. The proteins of the present invention can alsobe administered in combination with other cytokines such as IL-3, -6 and-11; stem cell factor; erythropoietin; G-CSF and GM-CSF. Within regimensof combination therapy, daily doses of other cytokines will in generalbe: EPO, 150 U/kg; GM-CSF, 5-15 lg/kg; IL-3, 1-5 lg/kg; and G-CSF, 1-25lg/kg. Combination therapy with EPO, for example, is indicated in anemicpatients with low EPO levels.

The proteins of the present invention can also be used ex vivo, such asin autologous marrow culture or liver cultures. For example, briefly,bone marrow is removed from a patient prior to chemotherapy and treatedwith ZCYTO18, optionally in combination with one or more othercytokines. The treated marrow is then returned to the patient afterchemotherapy to speed the recovery of the marrow. In addition, theproteins of the present invention can also be used for the ex vivoexpansion of marrow or peripheral blood progenitor (PBPC) cells. Priorto chemotherapy treatment, marrow can be stimulated with stem cellfactor (SCF) or G-CSF to release early progenitor cells into peripheralcirculation. These progenitors can be collected and concentrated fromperipheral blood and then treated in culture with ZCYTO18, optionally incombination with one or more other cytokines, including but not limitedto SCF, G-CSF, IL-3, GM-CSF, IL-6 or IL-11, to differentiate andproliferate into high-density megakaryocyte cultures, which can then bereturned to the patient following high-dose chemotherapy. Such ex vivouses are especially desirable in the instance that systemicadministration is not tolerated by a patient. Thus the present inventionto provide methods for stimulating the production of platelets andneutrophils in mammals including humans. The invention provides methodsfor stimulating platelet and neutrophil production in a mammal, ex vivotissue sample, or cell cultures. The methods comprise administering to amammal, ex vivo tissue sample, or cell culture a therapeuticallyeffective amount of a hematopoietic protein selected from the groupconsisting of (a) proteins comprising the sequence of amino acids of SEQID NO:3 from amino acid residue 22 to amino acid residue 167; (b)allelic variants of (a); and (d) species homologs of (a) or (b), whereinthe protein stimulates proliferation or differentiation of myeloid orlymphoid precursors, or the production of platelets, in combination witha pharmaceutically acceptable vehicle.

Moreover, the increase of platelets and neutrophils is desirable at awound site not only in patients with blood diseases or undergoingchemotherapy as described above, but under normal conditions. Apolypeptide such as ZCYTO18, that increases platelet levels in vivo, canbe used in topological formulations including gels, meshes, poultices,liquids, and the like to aid in the healing of common cuts, burns,lacerations, abrasions, and the like. Moreover, such applications can beapplied in any instance where the healing of skin, muscle, or the likeis desired, even internally, such as after surgery.

The proteins of the present invention are also valuable tools for the invitro study of the differentiation and development of hematopoieticcells, such as for elucidating the mechanisms of cell differentiationand for determining the lineages of mature cells, and may also findutility as proliferative agents in cell culture.

Differentiation is a progressive and dynamic process, beginning withpluripotent stem cells and ending with terminally differentiated cells.Pluripotent stem cells that can regenerate without commitment to alineage express a set of differentiation markers that are lost whencommitment to a cell lineage is made. Progenitor cells express a set ofdifferentiation markers that may or may not continue to be expressed asthe cells progress down the cell lineage pathway toward maturation.Differentiation markers that are expressed exclusively by mature cellsare usually functional properties such as cell products, enzymes toproduce cell products, and receptors. The stage of a cell population'sdifferentiation is monitored by identification of markers present in thecell population. Myocytes, osteoblasts, adipocytes, chrondrocytes,fibroblasts and reticular cells are believed to originate from a commonmesenchymal stem cell (Owen et al., Ciba Fdn. Symp. 136:42-46, 1988).Markers for mesenchymal stem cells have not been well identified (Owenet al., J. of Cell Sci. 87:731-738, 1987), so identification is usuallymade at the progenitor and mature cell stages. The novel polypeptides ofthe present invention may be useful for studies to isolate mesenchymalstem cells and myocyte or other progenitor cells, both in vivo and exvivo.

There is evidence to suggest that factors that stimulate specific celltypes down a pathway towards terminal differentiation ordedifferentiation affect the entire cell population originating from acommon precursor or stem cell. Thus, the present invention includesstimulating or inhibiting the proliferation of myocytes, smooth musclecells, osteoblasts, adipocytes, chrondrocytes, neuronal and endothelialcells. Molecules of the present invention for example, may whilestimulating proliferation or differentiation of cardiac myocytes,inhibit proliferation or differentiation of adipocytes, by virtue of theaffect on their common precursor/stem cells. Thus molecules of thepresent invention may have use in inhibiting chondrosarcomas,atherosclerosis, restenosis and obesity.

Assays measuring differentiation include, for example, measuring cellmarkers associated with stage-specific expression of a tissue, enzymaticactivity, functional activity or morphological changes (Watt, FASEB,5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes, Adv.Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989; all incorporatedherein by reference). Alternatively, ZCYTO18 polypeptide itself canserve as an additional cell-surface or secreted marker associated withstage-specific expression of a tissue. As such, direct measurement ofZCYTO18 polypeptide, or its loss of expression in a tissue as itdifferentiates, can serve as a marker for differentiation of tissues.

Similarly, direct measurement of ZCYTO18 polypeptide, or its loss ofexpression in a tissue can be determined in a tissue or cells as theyundergo tumor progression. Increases in invasiveness and motility ofcells, or the gain or loss of expression of ZCYTO18 in a pre-cancerousor cancerous condition, in comparison to normal tissue, can serve as adiagnostic for transformation, invasion and metastasis in tumorprogression. As such, knowledge of a tumor's stage of progression ormetastasis will aid the physician in choosing the most proper therapy,or aggressiveness of treatment, for a given individual cancer patient.Methods of measuring gain and loss of expression (of either mRNA orprotein) are well known in the art and described herein and can beapplied to ZCYTO18 expression. For example, appearance or disappearanceof polypeptides that regulate cell motility can be used to aid diagnosisand prognosis of prostate cancer (Banyard, J. and Zetter, B. R., Cancerand Metast. Rev. 17:449-458, 1999). As an effector of cell motility,ZCYTO18 gain or loss of expression may serve as a diagnostic forprostate and other cancers.

Moreover, the activity and effect of ZCYTO18 on tumor progression andmetastasis can be measured in vivo. Several syngeneic mouse models havebeen developed to study the influence of polypeptides, compounds orother treatments on tumor progression. In these models, tumor cellspassaged in culture are implanted into mice of the same strain as thetumor donor. The cells will develop into tumors having similarcharacteristics in the recipient mice, and metastasis will also occur insome of the models. Appropriate tumor models for our studies include theLewis lung carcinoma (ATCC No. CRL-1642) and B16 melanoma (ATCC No.CRL-6323), amongst others. These are both commonly used tumor lines,syngeneic to the C57BL6 mouse, that are readily cultured and manipulatedin vitro. Tumors resulting from implantation of either of these celllines are capable of metastasis to the lung in C57BL6 mice. The Lewislung carcinoma model has recently been used in mice to identify aninhibitor of angiogenesis (O′Reilly M S, et al. Cell 79: 315-328, 1994).C57BL6/J mice are treated with an experimental agent either throughdaily injection of recombinant protein, agonist or antagonist or a onetime injection of recombinant adenovirus. Three days following thistreatment, 10⁵ to 10⁶ cells are implanted under the dorsal skin.Alternatively, the cells themselves may be infected with recombinantadenovirus, such as one expressing ZCYTO18, before implantation so thatthe protein is synthesized at the tumor site or intracellularly, ratherthan systemically. The mice normally develop visible tumors within 5days. The tumors are allowed to grow for a period of up to 3 weeks,during which time they may reach a size of 1500-1800 mm³ in the controltreated group. Tumor size and body weight are carefully monitoredthroughout the experiment. At the time of sacrifice, the tumor isremoved and weighed along with the lungs and the liver. The lung weighthas been shown to correlate well with metastatic tumor burden. As anadditional measure, lung surface metastases are counted. The resectedtumor, lungs and liver are prepared for histopathological examination,immunohistochemistry, and in situ hybridization, using methods known inthe art and described herein. The influence of the expressed polypeptidein question, e.g., ZCYTO18, on the ability of the tumor to recruitvasculature and undergo metastasis can thus be assessed. In addition,aside from using adenovirus, the implanted cells can be transientlytransfected with ZCYTO18. Use of stable ZCYTO18 transfectants as well asuse of induceable promoters to activate ZCYTO18 expression in vivo areknown in the art and can be used in this system to assess ZCYTO18induction of metastasis. Moreover, purified ZCYTO18 or ZCYTO18conditioned media can be directly injected in to this mouse model, andhence be used in this system. For general reference see, O'Reilly M S,et al. Cell 79:315-328, 1994; and Rusciano D, et al. Murine Models ofLiver Metastasis. Invasion Metastasis 14:349-361, 1995.

Polynucleotides encoding ZCYTO18 polypeptides are useful within genetherapy applications where it is desired to increase or inhibit ZCYTO18activity. If a mammal has a mutated or absent ZCYTO18 gene, the ZCYTO18gene can be introduced into the cells of the mammal. In one embodiment,a gene encoding a ZCYTO18 polypeptide is introduced in vivo in a viralvector. Such vectors include an attenuated or defective DNA virus, suchas, but not limited to, herpes simplex virus (HSV), papillomavirus,Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), andthe like. Defective viruses, which entirely or almost entirely lackviral genes, are preferred. A defective virus is not infective afterintroduction into a cell. Use of defective viral vectors allows foradministration to cells in a specific, localized area, without concernthat the vector can infect other cells. Examples of particular vectorsinclude, but are not limited to, a defective herpes simplex virus 1(HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci. 2:320-30, 1991);an attenuated adenovirus vector, such as the vector described byStratford-Perricaudet et al., J. Clin. Invest. 90:626-30, 1992; and adefective adeno-associated virus vector (Samulski et al., J. Virol.61:3096-101, 1987; Samulski et al., J. Virol. 63:3822-8, 1989).

In another embodiment, a ZCYTO18 gene can be introduced in a retroviralvector, e.g., as described in Anderson et al., U.S. Pat. No. 5,399,346;Mann et al. Cell 33:153, 1983; Temin et al., U.S. Pat. No. 4,650,764;Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J. Virol.62:1120, 1988; Temin et al., U.S. Pat. No. 5,124,263; InternationalPatent Publication No. WO 95/07358, published Mar. 16, 1995 by Doughertyet al.; and Kuo et al., Blood 82:845, 1993. Alternatively, the vectorcan be introduced by lipofection in vivo using liposomes. Syntheticcationic lipids can be used to prepare liposomes for in vivotransfection of a gene encoding a marker (Feigner et al., Proc. Natl.Acad. Sci. USA 84:7413-7, 1987; Mackey et al., Proc. Natl. Acad. Sci.USA 85:8027-31, 1988). The use of lipofection to introduce exogenousgenes into specific organs in vivo has certain practical advantages.Molecular targeting of liposomes to specific cells represents one areaof benefit. More particularly, directing transfection to particularcells represents one area of benefit. For instance, directingtransfection to particular cell types would be particularly advantageousin a tissue with cellular heterogeneity, such as the pancreas, liver,kidney, and brain. Lipids may be chemically coupled to other moleculesfor the purpose of targeting. Targeted peptides (e.g., hormones orneurotransmitters), proteins such as antibodies, or non-peptidemolecules can be coupled to liposomes chemically.

It is possible to remove the target cells from the body; to introducethe vector as a naked DNA plasmid; and then to re-implant thetransformed cells into the body. Naked DNA vectors for gene therapy canbe introduced into the desired host cells by methods known in the art,e.g., transfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a gene gunor use of a DNA vector transporter. See, e.g., Wu et al., J. Biol. Chem.267:963-7, 1992; Wu et al., J. Biol. Chem. 263:14621-4, 1988.

Antisense methodology can be used to inhibit ZCYTO18 gene transcription,such as to inhibit cell proliferation in vivo. Polynucleotides that arecomplementary to a segment of a ZCYTO18-encoding polynucleotide (e.g., apolynucleotide as set froth in SEQ ID NO:1) are designed to bind toZCYTO18-encoding mRNA and to inhibit translation of such mRNA. Suchantisense polynucleotides are used to inhibit expression of ZCYTO18polypeptide-encoding genes in cell culture or in a subject.

The present invention also provides reagents which will find use indiagnostic applications. For example, the ZCYTO18 gene, a probecomprising ZCYTO18 DNA or RNA or a subsequence thereof can be used todetermine if the ZCYTO18 gene is present on a human chromosome, such aschromosome 12, or if a mutation has occurred. Based on annotation of afragment of human genomic DNA containing a part of ZCYTO18 genomic DNA(Genbank Accession No. AC007458), ZCYTO18 is located at the 12q15 regionof chromosome 12. Detectable chromosomal aberrations at the ZCYTO18 genelocus include, but are not limited to, aneuploidy, gene copy numberchanges, loss of heterogeneity (LOH), translocations, insertions,deletions, restriction site changes and rearrangements. Such aberrationscan be detected using polynucleotides of the present invention byemploying molecular genetic techniques, such as restriction fragmentlength polymorphism (RFLP) analysis, short tandem repeat (STR) analysisemploying PCR techniques, and other genetic linkage analysis techniquesknown in the art (Sambrook et al., ibid.; Ausubel et. al., ibid.;Marian, Chest 108:255-65, 1995).

The precise knowledge of a gene's position can be useful for a number ofpurposes, including: 1) determining if a sequence is part of an existingcontig and obtaining additional surrounding genetic sequences in variousforms, such as YACs, BACs or cDNA clones; 2) providing a possiblecandidate gene for an inheritable disease which shows linkage to thesame chromosomal region; and 3) cross-referencing model organisms, suchas mouse, which may aid in determining what function a particular genemight have.

ZCYTO18 is located at the 12q15 region of chromosome 12. Another T-cellexpressed cytokine, interferon-gamma (IFN-γ) maps near this locus(12q14), suggesting that the 12q14-15 locus is an important region forT-cell expressed cytokines. Moreover, mutations in IFN-γ are associatedwith immunodeficiency (See, e.g., Tzoneva, M. et al., Clin. Genet.33:454-456, 1988). Mutations in ZCYTO18, are likely to cause humandisease as well, such as immunodeficiency, autoimmune disease, lymphoidcell cancers, or other immune dysfunction. Moreover, there are severalgenes that map to the ZCYTO18 locus that are associated with humandisease states, such as cancer. 12q13-q15 region is involved in avariety of malignant and benign solid tumors (including salivaryadenomas and uterine leiomyomas), with 12q15 as a common break point.Moreover, the high mobility group protein isoform I-C(HMGIC) maps to12q15 and is involved in benign lipomas and other tumors. As ZCYTO18maps to 12q15 as well, there can be an association between loss ofZCYTO18 function and tumor formation or progression. Moreover,translocations in 12q13-15 are prevalent in soft tissue tumors, multiplelipomatosis and malignant mixoid liposarcoma. ZCYTO18 polynucleotideprobes can be used to detect abnormalities or genotypes associated withthese cancer susceptibility markers. Because there is abundant evidencefor cancer resulting from mutations in the 12q15 region, and ZCYTO18also maps to this chromosomal locus, mutations in ZCYTO18 may also bedirectly involved in or associated with cancers, such as lymphoid cellcancers or other tumors.

A diagnostic could assist physicians in determining the type of diseaseand appropriate associated therapy, or assistance in genetic counseling.As such, the inventive anti-ZCYTO18 antibodies, polynucleotides, andpolypeptides can be used for the detection of ZCYTO18 polypeptide, mRNAor anti-ZCYTO18 antibodies, thus serving as markers and be directly usedfor detecting or genetic diseases or cancers, as described herein, usingmethods known in the art and described herein. Further, ZCYTO18polynucleotide probes can be used to detect abnormalities or genotypesassociated with chromosome 12q15 deletions and translocations associatedwith human diseases, such as multiple lipomatosis and malignant mixoidliposarcoma (above), or other translocations involved with malignantprogression of tumors or other 12q15 mutations, which are expected to beinvolved in chromosome rearrangements in malignancy; or in othercancers. Similarly, ZCYTO18 polynucleotide probes can be used to detectabnormalities or genotypes associated with chromosome 12q15 trisomy andchromosome loss associated with human diseases or spontaneous abortion.Moreover, amongst other genetic loci, those for Scapuloperoneal spinalmuscular atrophy (12q13.3-q15), mucopolysaccaridosis (12q14),pseudo-vitamin D deficiency Rickets as a result of mutation inCytochrome CYP27B1 (12q14) and others, all manifest themselves in humandisease states as well as map to this region of the human genome. All ofthese serve as possible candidate genes for an inheritable disease whichshow linkage to the same chromosomal region as the ZCYTO18 gene. Thus,ZCYTO18 polynucleotide probes can be used to detect abnormalities orgenotypes associated with these defects.

As discussed above, defects in the ZCYTO18 gene itself may result in aheritable human disease state. Molecules of the present invention, suchas the polypeptides, antagonists, agonists, polynucleotides andantibodies of the present invention would aid in the detection,diagnosis prevention, and treatment associated with a ZCYTO18 geneticdefect. In addition, ZCYTO18 polynucleotide probes can be used to detectallelic differences between diseased or non-diseased individuals at theZCYTO18 chromosomal locus. As such, the ZCYTO18 sequences can be used asdiagnostics in forensic DNA profiling.

In general, the diagnostic methods used in genetic linkage analysis, todetect a genetic abnormality or aberration in a patient, are known inthe art. Analytical probes will be generally at least 20 nt in length,although somewhat shorter probes can be used (e.g., 14-17 nt). PCRprimers are at least 5 nt in length, preferably 15 or more, morepreferably 20-30 nt. For gross analysis of genes, or chromosomal DNA, aZCYTO18 polynucleotide probe may comprise an entire exon or more. Exonsare readily determined by one of skill in the art by comparing ZCYTO18sequences (SEQ ID NO:1) with the human genomic DNA for ZCYTO18 (GenbankAccession No. AC007458). In general, the diagnostic methods used ingenetic linkage analysis, to detect a genetic abnormality or aberrationin a patient, are known in the art. Most diagnostic methods comprise thesteps of (a) obtaining a genetic sample from a potentially diseasedpatient, diseased patient or potential non-diseased carrier of arecessive disease allele; (b) producing a first reaction product byincubating the genetic sample with a ZCYTO18 polynucleotide probewherein the polynucleotide will hybridize to complementarypolynucleotide sequence, such as in RFLP analysis or by incubating thegenetic sample with sense and antisense primers in a PCR reaction underappropriate PCR reaction conditions; (iii) Visualizing the firstreaction product by gel electrophoresis and/or other known method suchas visualizing the first reaction product with a ZCYTO18 polynucleotideprobe wherein the polynucleotide will hybridize to the complementarypolynucleotide sequence of the first reaction; and (iv) comparing thevisualized first reaction product to a second control reaction productof a genetic sample from wild type patient. A difference between thefirst reaction product and the control reaction product is indicative ofa genetic abnormality in the diseased or potentially diseased patient,or the presence of a heterozygous recessive carrier phenotype for anon-diseased patient, or the presence of a genetic defect in a tumorfrom a diseased patient, or the presence of a genetic abnormality in afetus or pre-implantation embryo. For example, a difference inrestriction fragment pattern, length of PCR products, length ofrepetitive sequences at the ZCYTO18 genetic locus, and the like, areindicative of a genetic abnormality, genetic aberration, or allelicdifference in comparison to the normal wild type control. Controls canbe from unaffected family members, or unrelated individuals, dependingon the test and availability of samples. Genetic samples for use withinthe present invention include genomic DNA, mRNA, and cDNA isolated formany tissue or other biological sample from a patient, such as but notlimited to, blood, saliva, semen, embryonic cells, amniotic fluid, andthe like. The polynucleotide probe or primer can be RNA or DNA, and willcomprise a portion of SEQ ID NO:1, the complement of SEQ ID NO:1, or anRNA equivalent thereof. Such methods of showing genetic linkage analysisto human disease phenotypes are well known in the art. For reference toPCR based methods in diagnostics see see, generally, Mathew (ed.),Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), White(ed.), PCR Protocols: Current Methods and Applications (Humana Press,Inc. 1993), Cotter (ed.), Molecular Diagnosis of Cancer (Humana Press,Inc. 1996), Hanausek and Walaszek (eds.), Tumor Marker Protocols (HumanaPress, Inc. 1998), Lo (ed.), Clinical Applications of PCR (Humana Press,Inc. 1998), and Meltzer (ed.), PCR in Bioanalysis (Humana Press, Inc.1998)).

Aberrations associated with the ZCYTO18 locus can be detected usingnucleic acid molecules of the present invention by employing standardmethods for direct mutation analysis, such as restriction fragmentlength polymorphism analysis, short tandem repeat analysis employing PCRtechniques, amplification-refractory mutation system analysis,single-strand conformation polymorphism detection, RNase cleavagemethods, denaturing gradient gel electrophoresis, fluorescence-assistedmismatch analysis, and other genetic analysis techniques known in theart (see, for example, Mathew (ed.), Protocols in Human MolecularGenetics (Humana Press, Inc. 1991), Marian, Chest 108:255 (1995),Coleman and Tsongalis, Molecular Diagnostics (Human Press, Inc. 1996),Elles (ed.) Molecular Diagnosis of Genetic Diseases (Humana Press, Inc.1996), Landegren (ed.), Laboratory Protocols for Mutation Detection(Oxford University Press 1996), Birren et al. (eds.), Genome Analysis,Vol. 2: Detecting Genes (Cold Spring Harbor Laboratory Press 1998),Dracopoli et al. (eds.), Current Protocols in Human Genetics (John Wiley& Sons 1998), and Richards and Ward, “Molecular Diagnostic Testing,” inPrinciples of Molecular Medicine, pages 83-88 (Humana Press, Inc.1998)). Direct analysis of an ZCYTO18 gene for a mutation can beperformed using a subject's genomic DNA. Methods for amplifying genomicDNA, obtained for example from peripheral blood lymphocytes, arewell-known to those of skill in the art (see, for example, Dracopoli etal. (eds.), Current Protocols in Human Genetics, at pages 7.1.6 to 7.1.7(John Wiley & Sons 1998)).

Mice engineered to express the ZCYTO18 gene, referred to as “transgenicmice,” and mice that exhibit a complete absence of ZCYTO18 genefunction, referred to as “knockout mice,” may also be generated(Snouwaert et al., Science 257:1083, 1992; Lowell et al., Nature366:740-42, 1993; Capecchi, M. R., Science 244: 1288-1292, 1989;Palmiter, R. D. et al. Annu Rev Genet. 20: 465-499, 1986). For example,transgenic mice that over-express ZCYTO18, either ubiquitously or undera tissue-specific or tissue-restricted promoter can be used to askwhether over-expression causes a phenotype. For example, over-expressionof a wild-type ZCYTO18 polypeptide, polypeptide fragment or a mutantthereof may alter normal cellular processes, resulting in a phenotypethat identifies a tissue in which ZCYTO18 expression is functionallyrelevant and may indicate a therapeutic target for the ZCYTO18, itsagonists or antagonists. For example, a preferred transgenic mouse toengineer is one that over-expresses the mature ZCYTO18 polypeptide(amino acid residues 23 (Pro) to 167 (Ile) of SEQ ID NO:3). Moreover,such over-expression may result in a phenotype that shows similaritywith human diseases. Similarly, knockout ZCYTO18 mice can be used todetermine where ZCYTO18 is absolutely required in vivo. The phenotype ofknockout mice is predictive of the in vivo effects of that a ZCYTO18antagonist, such as those described herein, may have. The human or mouseZCYTO18 cDNA can be used to generate knockout mice. These mice may beemployed to study the ZCYTO18 gene and the protein encoded thereby in anin vivo system, and can be used as in vivo models for correspondinghuman diseases. Moreover, transgenic mice expression of ZCYTO18antisense polynucleotides or ribozymes directed against ZCYTO18,described herein, can be used analogously to transgenic mice describedabove. Studies may be carried out by administration of purified ZCYTO18protein, as well.

For pharmaceutical use, the proteins of the present invention areformulated for parenteral, particularly intravenous or subcutaneous,delivery according to conventional methods. Intravenous administrationwill be by bolus injection or infusion over a typical period of one toseveral hours. In general, pharmaceutical formulations will include aZCYTO18 protein in combination with a pharmaceutically acceptablevehicle, such as saline, buffered saline, 5% dextrose in water or thelike. Formulations may further include one or more excipients,preservatives, solubilizers, buffering agents, albumin to preventprotein loss on vial surfaces, etc. Methods of formulation are wellknown in the art and are disclosed, for example, in Remington: TheScience and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co.,Easton, Pa., 19th ed., 1995. Therapeutic doses will generally be in therange of 0.1 to 100 μg/kg of patient weight per day, preferably 0.5-20mg/kg per day, with the exact dose determined by the clinician accordingto accepted standards, taking into account the nature and severity ofthe condition to be treated, patient traits, etc. Determination of doseis within the level of ordinary skill in the art. The proteins may beadministered for acute treatment, over one week or less, often over aperiod of one to three days or may be used in chronic treatment, overseveral months or years. In general, a therapeutically effective amountof ZCYTO18 is an amount sufficient to produce a clinically significantchange in hematopoietic or immune function.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Using an EST Sequence to Identify and Clone ZCYTO18

Novel ZCYTO18 encoding polynucleotides and polypeptides of the presentinvention were initially identified by querying an EST database forsequences homologous to conserved motifs within the cytokine family. Aprimary expressed sequence tag (EST) from a human T-lymphocyte cDNAlibrary was identified.

An initial partial sequence was obtained from the sequencing of the EST(INC4345486). Additional 5′ sequence was obtained from sequencing thecDNA fragment obtained by PCR from the Northern Analysis (Example 2,below) and by further PCR using oligonucleotides ZC25,840 (SEQ ID NO:5)and ZC25,841 (SEQ ID NO:6) in a PCR using human mixed lymphocytereaction (MLR) cDNA. Thermocycler conditions were as described inExample 2 below. The resulting 1082 by full length sequence is disclosedin SEQ ID NO:1 and the corresponding amino acid sequence is shown in SEQID NO:2 and SEQ ID NO:3. The full length novel cytokine was designatedZCYTO18.

Example 2 Zcyto18 Tissue Distribution

Northerns were performed using Human Multiple Tissue Blots (MTN1, MTN2and MTN3) from Clontech (Palo Alto, Calif.) to determine the tissuedistribution of human ZCYTO18. A 237 by cDNA probe was obtained usingthe PCR. Oligonucleotides ZC25,838 (SEQ ID NO:7) and ZC25,839 (SEQ IDNO:8) were used as primers. Marathon cDNA, synthesized in-house usingMarathon cDNA Kit (Clontech) and protocol, was used as a template. Thefollowing human tissue specific cDNAs were also used: lymph node, bonemarrow, CD4+, CD8+, spleen, and MLR, along with human genomic DNA(Clontech). Thermocycler conditions were as follows: one cycle at 94° C.for 2 min.; 35 cycles of 94° C. for 15 sec., 62° C. for 20 sec., and 72°C. for 30 sec.; one cycle at 72° C. for 7 min.; followed by a 4° C.hold. The correct predicted band size (237 bp) was observed on a 4%agarose gel in CD4+ and MLR reactions, along with the genomic DNAreaction. A band was excised and purified using a Gel Extraction Kit(Qiagen, Chatsworth, Calif.) according to manufacturer's instructions.The cDNA was radioactively labeled using a Rediprime II DNA labeling kit(Amersham, Arlington Heights, Ill.) according to the manufacturer'sspecifications. The probe was purified using a NUCTRAP push column(Stratagene Cloning Systems, La Jolla, Calif.). EXPRESSHYB (Clontech,Palo Alto, Calif.) solution was used for prehybridization and as ahybridizing solution. Hybridization took place overnight at 55° C.,using 2×10⁶ cpm/ml labeled probe. The blots were then washed in 2×SSCand 0.1% SDS at room temperature, then with 2×SSC and 0.1% SDS at 65°C., followed by a wash in 0.1×SSC and 0.1% SDS at 65° C. The blots wereexposed 5 days to Biomax MS film (Kodak, Rochester, N.Y.). No transcriptsignals were observed on the MTN blots after development.

A RNA Master Dot Blot (Clontech) that contained RNAs from varioustissues that were normalized to 8 housekeeping genes was also probed andhybridized as described above. A signal was observed in genomic DNA.While a faint signal in lymph node and very faint signals in fetalliver, skeletal muscle, and placenta were observed it was inconclusivewhether these signals were significantly above background.

Example 3 Identification of Cells Expressing ZCYTO18 Using RT-PCR

Specific human cell types were isolated and screened for ZCYTO18expression by RT-PCR. B-cells were isolated from fresh human tonsils bymechanical disruption through 100 μm nylon cell strainers (BectonDickinson Biosciences, Franklin Lakes, N.J.). The B-cell suspensionswere enriched for CD19+ B-cells by positive selection with VarioMACS VS+magnetic column and CD19 microbeads (Miltenyi Biotec, Auburn, Calif.) asper manufacturer's instructions. T-cells were isolated from humanapheresed blood samples. CD3+ T-cells were purified by CD3 microbeadVarioMACS positive selection and monocytes were purified by VarioMACSnegative selection columns (Miltenyi) as per manufacturer'sinstructions. Samples from each population were stained and analyzed byfluorescent antibody cell sorting (FACS) (Bectin Dickinson, San Jose,Calif.) analysis to determine the percent enrichment and resultingyields. CD19+ B-cells were approximately 96% purified, CD3+ T-cells wereapproximately 95% purified, and monocytes were approximately 96%purified.

RNA was prepared, using a standard method in the art, from all threecell types that were either resting or activated. RNA was isolated fromresting cells directly from the column preparations above. The CD19+ andCD3+ cells were activated by culturing at 500,000 cells/ml in RPMI+10%FBS containing PMA 5 ng/ml (Calbiochem, La Jolla, Calif.) and Ionomycin0.5 ug/ml (Calbiochem) for 4 and 24 hours. The monocytes were activatedby culturing in RPMI+10% FBS containing LPS 10 ng/ml (Sigma St. LouisMo.) and rhIFN-g 10 ng/ml (R&D, Minneapolis, Minn.) for 24 hours. Cellswere harvested and washed in PBS. RNA was prepared from the cell pelletsusing RNeasy Midiprep™ Kit (Qiagen, Valencia, Calif.) as permanufacturer's instructions and first strand cDNA synthesis wasgenerated with Superscript II™ Kit (GIBCO BRL, Grand Island, N.Y.) asper manufacturers protocol.

Oligos ZC25,838 (SEQ ID NO:7) and ZC25,840 (SEQ ID NO:5) were used in aPCR reaction to screen the above described samples for a 473 by fragmentcorresponding to ZCYTO18 message. PCR amplification was performed withTaq Polymerase (BRL Grand Island N.Y.), and reaction conditions asfollows: 35 cycles of 94° C. for 15 sec., 62° C. for 20 sec., 72° C. for30 sec.; 1 cycle at 72° C. for 7 min.; and 4° C. soak. 5 ul of each 50μl reaction volume was run on a 0.9% agarose 0.5XTBE gel to identifyresultant products. Table 5 below describes the results. PCR productswere scored as (−) for no product, (+) for expected PCR product visible,(++) increased presence of PCR product and (+++) being the strongestsignal.

TABLE 5 Cells expressing ZCYTO18 using RT-PCR cDNA Source Activation PCRProduct CD3+ cells  0 hr resting + 4-hr activated +++ CD19+ cells  4 hractivated ++ 24 hr activated + Monocytes 24 hr activated −

These results indicated that ZCYTO18 message is present in resting CD3+T-cells and increases with mitogenic activation. It also appears to beexpressed by 4-hr activated human CD19+ B-cells and reduced inexpression in 24 hr activated B-cells. There was no apparent message inactivated monocytes.

Example 4 Identification of hZCYTO18 Message in an Activated T-CellLibrary

A. The Vector for CD3+Selected Library Construction

The vector for CD3+ selected library construction was pZP7NX. The pZP7NXvector was previously constructed as follows: The coding region for theDHFR selective marker in vector pZP7 was removed by DNA digestion withNcoI and PstI restriction enzymes (Boehringer Mannheim). The digestedDNA was run on 1% agarose gel, cut out and gel purified using the QiagenGel Extraction Kit (Qiagen) as per manufacturer's instructions. A DNAfragment representing the coding region of Zeocin selective marker wasamplified by PCR method with primers ZC13,946 (SEQ ID NO:9) and ZC13,945(SEQ ID NO:10), and pZeoSV2(+) as a template. There are additional PstIand BclI restriction sites in primer ZC13,946 (SEQ ID NO:9), andadditional NcoI and SfuI sites in primer ZC13,945 (SEQ ID NO:10). ThePCR fragment was cut with PstI and NcoI restriction enzymes and clonedinto pZP7 vector prepared by cleaving with the same two enzymes andsubsequent gel purification. This vector was named pZP7Z. Then theZeocin coding region was removed by DNA digestion of vector pZP7Z withBclI and SfuI restriction enzymes. The digested DNA was run on 1%agarose gel, cut out and gel purified, and then ligated with a DNAfragment of Neomycin coding region cut from pZem228 vector with the samerestriction enzymes (BclI and SfuI).

This new vector was named pZP7N, in which the coding region for DHFRselective marker was replaced by the coding region for a Neomycinselective marker from vector pZem228. A stuffer fragment including anXho1 site was added to pZP7N to create a vector suitable for highefficiency directional cloning of cDNA; this new vector was calledpZP7NX. To prepare the vector for cDNA, 20 μg of pZP7NX was digestedwith 20 units of EcoR1 (Life Technologies Gaithersberg, Md.) and 20units of Xho1 (Boehringer Mannheim Indianapolis, Ind.) for 5 hours at37° C., then 68° C. for 15 minutes. The digest was then run on a 0.8%low melt agarose 1×TAE gel to separate the stuffer from the vector. Thevector band was excised and digested with “beta-Agarase” (New EnglandBiolabs, Beverly, Mass.) following the manufacturer's recommendations.After ethanol precipitation the digested vector was resuspended in waterto 45 ng/ml in preparation for ligation of CD3+ selected cDNA librarydescribed below.

B. Preparation of Primary Human Activated CD3+Selected Cell cDNA Library

Approximately 1.5×10⁸ primary human CD3+ selected cells stimulated inionomycin/PMA were isolated by centrifugation after culturing at 37° C.for 13 hours. Total RNA was isolated from the cell pellet using the“RNeasy Midi” kit from Qiagen, Inc. (Valencia, Calif.). mRNA wasisolated from 225 micrograms of total RNA using the “MPG mRNApurification kit” from CPG Inc. (Lincoln Park, N.J.). 3.4 micrograms ofmRNA was isolated and converted to double stranded cDNA using thefollowing procedure.

First strand cDNA from stimulated human CD3+ selected cells wassynthesized as follows. Nine μl Oligo d(T)-selected poly(A) CD3+ RNA ata concentration of 0.34 mg/μl and 1.0 μl of 1 μg/μl first strand primerZC18,698 (SEQ ID NO:11) containing an XhoI restriction site were mixedand heated at 65° C. for 4 minutes and cooled by chilling on ice. Firststrand cDNA synthesis was initiated by the addition of 9 μl of firststrand buffer (5× SUPERSCRIPT® buffer; Life Technologies), 4 μl of 100mM dithiothreitol and 2 μl of a deoxynucleotide triphosphate solutioncontaining 10 mM each of dATP, dGTP, dTTP and 5-methyl-dCTP (PharmaciaBiotech Inc.) to the RNA-primer mixture. The reaction mixture wasincubated at 45° C. for 4 minutes followed by the addition of 8 μl of200 U/μl SuperscriptII®, RNase H— reverse transcriptase (LifeTechnologies). The reaction was incubated at 45° C. for 45 minutesfollowed by an incubation ramp of 1° C. every 2 minutes to 50° C. wherethe reaction was held for 10 minutes. To denature any secondarystructure and allow for additional extension of the cDNA the reactionwas then heated to 70° C. for 2 minutes then dropped to 55° C. for 4minutes after which 2 μl of SuperscriptII® RT was added and incubated anadditional 15 minutes followed by a ramp up to 70° C. at 1 minute/1° C.Unincorporated nucleotides were removed from the cDNA by twiceprecipitating in the presence of 2 μg of glycogen carrier, 2.0 Mammonium acetate and 2.5 volume ethanol, followed by a 100 μl wash with70% ethanol. The cDNA was resuspended in 98 μl water for use in secondstrand synthesis.

Second strand synthesis was performed on the first strand cDNA underconditions that promoted first strand priming of second strand synthesisresulting in DNA hairpin formation. The second strand reaction contained98 μl of the first strand cDNA, 30 μl of 5× polymerase I buffer (100 mMTris: HCl, pH 7.5, 500 mM KCl, 25 mM MgCl2, 50 mM (NH₄)2SO4), 2 μl of100 mM dithiothreitol, 6 μl of a solution containing 10 mM of eachdeoxynucleotide triphosphate, 5 μl of 5 mM b-NAD, 1 μl of 3 U/μl E. coliDNA ligase (New England Biolabs Inc.) and 4 μl of 10 U/μl E. coli DNApolymerase I (New England Biolabs Inc.). The reaction was assembled atroom temperature and was incubated at room temperature for 2 minutesfollowed by the addition of 4 μl of 3.8 U/μl RNase H (LifeTechnologies). The reaction was incubated at 15° C. for two hoursfollowed by a 15 minute incubation at room temperature. 10 μl of 1M TRISpH7.4 was added to the reaction and extracted twice withphenol/chloroform and once with chloroform, the organic phases were thenback extracted with 50 μl of TE (10 mM TRIS pH 7.4, 1 mM EDTA), pooledwith the other aqueous and ethanol precipitated in the presence of 0.3 Msodium acetate. The pellet was washed with 100 μl 70% ethanol air driedand resuspended in 40 μl water.

The single-stranded DNA of the hairpin structure was cleaved using mungbean nuclease. The reaction mixture contained 40 μl of second strandcDNA, 5 μl of 10× mung bean nuclease buffer (Life technologies), 5 μl ofmung bean nuclease (Pharmacia Biotech Corp.) diluted to 1 U/μl in 1×mung bean nuclease buffer. The reaction was incubated at 37° C. for 45minutes. The reaction was terminated by the addition of 10 μl of 1 MTris: HCl, pH 7.4 followed by sequential phenol/chloroform andchloroform extractions as described above. Following the extractions,the cDNA was ethanol precipitated in the presence of 0.3 M sodiumacetate. The pellet was washed with 100 μl 70% ethanol air dried andresuspended in 38 μl water.

The resuspended cDNA was blunt-ended with T4 DNA polymerase. The cDNA,which was resuspended in 38 μl of water, was mixed with 12 μl 5× T4 DNApolymerase buffer (250 mM Tris:HCl, pH 8.0, 250 mM KCl, 25 mM MgC12), 2μl 0.1 M dithiothreitol, 6 μl of a solution containing 10 mM of eachdeoxynucleotide triphosphate and 2 μl of 1 U/μl T4 DNA polymerase(Boehringer Mannheim Corp.). After an incubation of 45 minutes at 15°C., the reaction was terminated by the addition of 30 μl TE followed bysequential phenol/chloroform and chloroform extractions and backextracted with 20 μl TE as described above. The DNA was ethanolprecipitated in the presence of 2 μl Pellet Paint™ (Novagen) carrier and0.3 M sodium acetate and was resuspended 11 μl of water.

Eco RI adapters were ligated onto the 5′ ends of the cDNA describedabove to enable cloning into an expression vector. 11 μl of cDNA and 4μl of 65 pmole/μl of Eco RI hemiphophorylated adaptor (Pharmacia BiotechCorp) were mixed with 5 μl 5× ligase buffer (Life Technologies), 2 μl of10 mM ATP and 3 μl of 1 U/μl T4 DNA ligase (Life Technologies), 1 μl 10×ligation buffer (Promega Corp), 9 μl water. The extra dilution with 1×buffer was to prevent the pellet paint from precipitating. The reactionwas incubated 9 hours in a water bath temperature ramp from 10° C. to22° C. over 9 hours, followed by 45 minutes at 25° C. The reaction wasterminated by incubation at 68° C. for 15 minutes.

To facilitate the directional cloning of the cDNA into an expressionvector, the cDNA was digested with XhoI, resulting in a cDNA having a 5′Eco RI cohesive end and a 3′ XhoI cohesive end. The XhoI restrictionsite at the 3′ end of the cDNA had been previously introduced using theZC18698 primer. Restriction enzyme digestion was carried out in areaction mixture containing 35 μl of the ligation mix described above, 6μl of 10× H buffer (Boehringer Mannheim Corp.), 1 μl of 2 mg/ml BSA(Biolabs Corp.), 17 μl water and 1.0 μl of 40 U/μl XhoI (BoehringerMannheim). Digestion was carried out at 37° C. for 1 hour. The reactionwas terminated by incubation at 68° C. for 15 minutes followed byethanol precipitation, washing drying as described above andresuspension in 30 μl water.

The resuspended cDNA was heated to 65° C. for 5 minutes and cooled onice, 4 μl of 5× gel loading dye (Research Genetics Corp.) was added, thecDNA was loaded onto a 0.8% low melt agarose 1×TAE gel (SEA PLAQUE GTG™low melt agarose; FMC Corp.) and electrophoresed. The contaminatingadapters and cDNA below 0.6 Kb in length were excised from the gel. Theelectrodes were reversed, molten agarose was added to fill in the wells,the buffer was changed and the cDNA was electrophoresed untilconcentrated near the lane origin. The area of the gel containing theconcentrated cDNA was excised and placed in a microfuge tube, and theagarose was melted by heating to 65° C. for 15 minutes. Followingequilibration of the sample to 45° C., 2 μl of 1 U/μl Beta-agarase I(Biolabs, Inc.) was added, and the mixture was incubated for 90 min. at45° C. to digest the agarose. After incubation, 1 tenth volume of 3 M Naacetate was added to the sample, and the mixture was incubated on icefor 15 minutes. The sample was centrifuged at 14,000×g for 15 minutes atroom temperature to remove undigested agarose, the cDNA was ethanolprecipitated, washed in 70% ethanol, air-dried and resuspended in 40 μlwater.

To determine the optimum ratio of cDNA to vector several ligations wereassembled and electroporated. Briefly, 2 μl of 5× T4 ligase buffer (LifeTechnologies), 1 μl of 10 mM ATP, 1 μl pZP7NX digested with EcoR1-Xho1,1 μl T4 DNA ligase diluted to 0.25u/μl (Life Technologies) water to 10μl and 0.5, 1, 2 or 3 μl of cDNA were mixed in 4 separate ligations,incubated at 22° C. for 4 hours, 68° C. for 20 minutes, sodiumacetate-ethanol precipitated, washed, dried and resuspended in 10 ll. Asingle microliter of each ligation was electroporated into 40 μl DH10bElectroMax™ electrocompetent bacteria (Life Technologies) using a 0.1 cmcuvette (Biorad) and a Genepulser, pulse controllerä (Biorad) set to 2.5KV, 25 lF, 200 ohms. These cells were immediately resuspended in 1 ml.SOC broth (Manniatis, et al. supra.) followed by 500 ll of 50%glycerol-SOC as a preservative. These “glycerol stocks” were frozen inseveral aliquots at −70° C. An aliquot of each was thawed and platedserially on LB-agar plates supplemented with ampicillin at 100 μg/ml.Colony numbers indicated that the optimum ratio of CD3+ cDNA to pZP7NXvector was 1 μl to 45 ng; such a ligation yielded 4.5 million primaryclones.

C. PCR Identification of Zcyto18 Message in Activated T-Cell Library

PCR was performed using oligos ZC25,838 (SEQ ID NO:7) and ZC25,840 (SEQID NO:5) to screen the above library for presence of a 473 by productcorresponding to ZCYTO18 clones. PCR amplification was performed withTaxi Polymerase (BRL Grand Island N.Y.), and conditions as follows: 30cycles of 94° C. for 15 sec., 62° C. 20 sec., 72° C. 30 sec.; 1 cycle at72° C. for 7 min.; and a 4° C. soak. 5 μl of each 50 μl reaction volumewas run on a 0.9% agarose 0.5×TBE gel to identify resultant products.Table 6 below describes the results. PCR products were scored as (−) forno product, (+) for expected PCR product visible, (++) increasedpresence of PCR product and (+++) being the strongest signal.

TABLE 6 Identification of ZCYTO18 message in activated T-Cell LibraryTemplate PCR Product  1 ng Activated Library +  10 ng Activated Library++ 100 ng Activated Library +++ 100 ng Vector Control − No TemplateControl −

These results indicate the presence of a ZCYTO18 cDNA clone andtherefore message in activated CD3+ T-cells.

Example 5 Southern Blot Analysis

Southern blots were performed using EVO Mammalian Group/EcoRI SouthernBlots (Quantum Biotechnologies, Inc., Montreal, Canada) to determine thepresence of orthologous ZCYTO18 sequences. A ZCYTO18 probe was generatedby labeling 25 ng of ZCYTO18 fragment, as described in Example 2, usingPrime-It II Random Primer labeling kit (Stratagene, La Jolla, Calif.).Hybridization was performed using Expresshyb (Clontech) with 5×10⁵cpm/ml probe and conditions of 65° C. overnight. Stringency washes wereperformed with 0.2×SSC, 0.1% SDS at 45° C. The blot was exposedovernight at −80° C. to X-ray film and analyzed.

Results showed a strong approximately 1 kb band in the human genomic DNAsample with weaker bands present at approximately 7 and 20 kb for murinegenomic DNA demonstrating the presence of a putative murine homolog forZCYTO18.

The mouse cDNA sequence was cloned using standard methods and is shownin SEQ ID NO:37, and corresponding polypeptides sequence shown in SEQ IDNO:38.

Example 6 Chromosomal Assignment and Placement of Zcyto18

Zcyto18 was mapped to chromosome 12 using the commercially availableversion of the “Stanford G3 Radiation Hybrid Mapping Panel” (ResearchGenetics, Inc., Huntsville, AL). The “Stanford G3 RH Panel” contains DNAfrom each of 83 radiation hybrid clones of the whole human genome, plustwo control DNAs (the RM donor and the A3 recipient).

For the mapping of Zcyto18 with the “Stanford G3 RH Panel”, 20 μlreactions were set up in a 96-well microtiter plate compatible for PCR(Stratagene, La Jolla, Calif.) and used in a “RoboCycler Gradient 96”thermal cycler (Stratagene). Each of the 85 PCR reactions consisted of 2μl 10× KlenTaq PCR reaction buffer (CLONTECH Laboratories, Inc., PaloAlto, Calif.), 1.6 μl dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City,Calif.), 1 μl sense primer, ZC 26,414 (SEQ ID NO:12), 1 antisenseprimer, ZC 26,415 (SEQ ID NO:13), 2 μl “RediLoad” (Research Genetics,Inc., Huntsville, Ala.), 0.4 μl 50× Advantage KlenTaq Polymerase Mix(Clontech Laboratories, Inc.), 25 ng of DNA from an individual hybridclone or control and distilled water for a total volume of 20 μl. Thereactions were overlaid with an equal amount of mineral oil and sealed.The PCR cycler conditions were as follows: an initial 1 cycle 5 minutedenaturation at 94° C., 35 cycles of a 45 seconds denaturation at 94°C., 45 seconds annealing at 66° C. and 1 minute AND 15 seconds extensionat 72° C., followed by a final 1 cycle extension of 7 minutes at 72° C.The reactions were separated by electrophoresis on a 2% agarose gel (EMScience, Gibbstown, N.J.) and visualized by staining with ethidiumbromide.

The results showed linkage of Zcyto18 to the chromosome 12 markerSHGC-17533 with a LOD score of >12 and at a distance of 0 cR_(—)10000from the marker. The use of surrounding genes and markers positionsZcyto18 in the 12q14-q24.3 chromosomal region.

Example 7 Construct for Generating CEE-Tagged Zcyto18

Oligonucleotides were designed to generate a PCR fragment containing theKozak sequence and the coding region for ZCYTO18, without its stopcodon. These oligonucleotides were designed with a KpnI site at the 5′end and a BamHI site at the 3′ end to facilitate cloning intopHZ200-CEE, our standard vector for mammalian expression of C-terminalGlu-Glu tagged (SEQ ID NO:14) proteins. The pHZ200 vector contains anMT-1 promoter.

PCR reactions were carried out using Turbo Pfu polymerase (Stratagene)to amplify a ZCYTO18 cDNA fragment. About 20 ng human ZCYTO18polynucleotide template (SEQ ID NO:1), and oligonucleotides ZC28590 (SEQID NO:16) and ZC28580 (SEQ ID NO:17) were used in the PCR reaction. PCRreaction conditions were as follows: 95° C. for 5 minutes; 30 cycles of95° C. for 60 seconds, 55° C. for 60 seconds, and 72° C. for 60 seconds;and 72° C. for 10 minutes; followed by a 4° C. hold. PCR products wereseparated by agarose gel electrophoresis and purified using a QiaQuick™(Qiagen) gel extraction kit. The isolated, approximately 600 bp, DNAfragment was digested with KpnI and BamHI (Boerhinger-Mannheim), gelpurified as above and ligated into pHZ200-CEE that was previouslydigested with KpnI and BamHI.

About one microliter of the ligation reaction was electroporated intoDH10B ElectroMax™ competent cells (GIBCO BRL, Gaithersburg, Md.)according to manufacturer's direction and plated onto LB platescontaining 100 μg/ml ampicillin, and incubated overnight. Colonies werepicked and screened by PCR using oligonucleotides ZC28,590 (SEQ IDNO:16) and ZC28,580 (SEQ ID NO:17), with PCR conditions as describedabove. Clones containing inserts were then sequenced to confirmerror-free ZCYTO18 inserts. Maxipreps of the correct pHZ200-ZCYTO18-CEEconstruct, as verified by sequence analysis, were performed.

Example 8 Transfection and Expression of Zcyto18-CEE Polypeptides

BHK 570 cells (ATCC No. CRL-10314), were plated at about 1×10⁶ cells/100mm culture dish in 6.4 ml of serum free (SF) DMEM media (DMEM, Gibco/BRLHigh Glucose) (Gibco BRL, Gaithersburg, Md.). The cells were transfectedwith an expression plasmid containing ZCYTO18-CEE described above(Example 7), using Lipofectin™ (Gibco BRL), in serum free (SF) DMEMaccording to manufacturer's instructions.

The cells were incubated at 37° C. for approximately five hours, then 10ml of DMEM/10% fetal bovine serum (FBS) (Hyclone, Logan, Utah) wasadded. The plates were incubated at 37° C., 5% CO₂, overnight and theDMEM/10% FBS media was replaced with selection media (5% FBS/DMEM with 1μM methotrexate (MTX)) the next day.

Approximately 7-10 days post-transfection, pools of cells or colonieswere mechanically picked to 12-well plates in one ml of 5% FCS/DMEM with5 μM MTX, then grown to confluence. Cells were then incubated in 5%FCS/DMEM with 10 μM MTX for at least 14 days. Conditioned media samplesfrom positive expressing clonal colonies and pools were then tested forexpression levels via SDS-PAGE and Western analysis. A high-expressingclonesor pools were picked and expanded for ample generation ofconditioned media for purification of the ZCYTO18-CEE expressed by thecells (Example 9).

Example 9 Purification of ZCYTO18-CEE from BHK 570 Cells

Unless otherwise noted, all operations were carried out at 4° C. Thefollowing procedure was used for purifying ZCYTO18 polypeptidecontaining C-terminal GluGlu (EE) tags (SEQ ID NO:14). A Proteaseinhibitor solution was added to the concentrated conditioned mediacontaining ZCYTO18-CEE (Example 8) to final concentrations of 2.5 mMethylenediaminetetraacetic acid (EDTA, Sigma Chemical Co. St. Louis,Mo.), 0.003 mM leupeptin (Boehringer-Mannheim, Indianapolis, Ind.),0.001 mM pepstatin (Boehringer-Mannheim) and 0.4 mM Pefabloc(Boehringer-Mannheim).

About 100 ml column of anti-EE G-Sepharose (prepared as described below)was poured in a Waters AP-5, 5 cm×10 cm glass column. The column wasflow packed and equilibrated on a BioCad Sprint (PerSeptive BioSystems,Framingham, Mass.) with phosphate buffered saline (PBS) pH 7.4. Theconcentrated conditioned media was 0.2 micron sterile filtered, pHadjusted to 7.4, then loaded on the column overnight with about 1ml/minute flow rate. The column was washed with 10 column volumes (CVs)of phosphate buffered saline (PBS, pH 7.4), then plug eluted with 200 mlof PBS (pH 6.0) containing 0.1 mg/ml EE peptide (Anaspec, San Jose,Calif.) at 5 ml/minute. The EE peptide used has the sequence EYMPME (SEQID NO:14). Five ml fractions were collected over the entire elutionchromatography and absorbance at 280 and 215 nM were monitored; the passthrough and wash pools were also saved and analyzed. The EE-polypeptideelution peak fractions were analyzed for the target protein via SDS-PAGESilver staining and Western Blotting with the anti-EE HRP conjugatedantibody. The polypeptide elution fractions of interest were pooled andconcentrated from 60 ml to 5.0 ml using a 10,000 Dalton molecular weightcutoff membrane spin concentrator (Millipore, Bedford, Mass.) accordingto the manufacturer's instructions.

To separate ZCYTO18-CEE polypeptide from free EE peptide and anycontaminating co-purifying proteins, the pooled concentrated fractionswere subjected to size exclusion chromatography on a 1.5×90 cm Sephadex5200 (Pharmacia, Piscataway, N.J.) column equilibrated and loaded in PBSat a flow rate of 1.0 ml/min using a BioCad Sprint. 1.5 ml fractionswere collected across the entire chromatography and the absorbance at280 and 215 nM were monitored. The peak fractions were characterized viaSDS-PAGE Silver staining, and only the most pure fractions were pooled.This material represented purified ZCYTO18-CEE polypeptide.

This purified material was finally subjected to a 4 ml ActiClean Etox(Sterogene) column to remove any remaining endotoxins. The sample waspassed over the PBS equilibrated gravity column four times then thecolumn was washed with a single 3 ml volume of PBS, which was pooledwith the “cleaned” sample. The material was then 0.2 micron sterilefiltered and stored at −80° C. until it was aliquoted.

On Western blotted, Coomassie Blue and Silver stained SDS-PAGE gels, theZCYTO18-CEE polypeptide was two major bands and two mionor bands. Theprotein concentration of the purified material was performed by BCAanalysis (Pierce, Rockford, Ill.) and the protein was aliquoted, andstored at −80° C. according to standard procedures. In a Western blotanalysis, all bands were immunoreactive with a rabbitanti-ZCYTO18-peptide antibody (Example 16). The 4 bands likely representdifferent glycosylated forms of the ZCYTO18 polypeptide.

To prepare anti-EE Sepharose, a 100 ml bed volume of protein G-Sepharose(Pharmacia, Piscataway, N.J.) was washed 3 times with 100 ml of PBScontaining 0.02% sodium azide using a 500 ml Nalgene 0.45 micron filterunit. The gel was washed with 6.0 volumes of 200 mM triethanolamine, pH8.2 (TEA, Sigma, St. Louis, Mo.), and an equal volume of EE antibodysolution containing 900 mg of antibody was added. After an overnightincubation at 4° C., unbound antibody was removed by washing the resinwith 5 volumes of 200 mM TEA as described above. The resin wasresuspended in 2 volumes of TEA, transferred to a suitable container,and dimethylpimilimidate-2HCl (Pierce, Rockford, Ill.) dissolved in TEA,was added to a final concentration of 36 mg/ml of protein G-Sepharosegel. The gel was rocked at room temperature for 45 min and the liquidwas removed using the filter unit as described above. Nonspecific siteson the gel were then blocked by incubating for 10 min at roomtemperature with 5 volumes of 20 mM ethanolamine in 200 mM TEA. The gelwas then washed with 5 volumes of PBS containing 0.02% sodium azide andstored in this solution at 4° C.

Example 10 Generation of Non-tagged ZCYTO18 Recombinant Adenovirus

The protein coding region of human ZCYTO18 (SEQ ID NO:1; SEQ ID NO:2)was amplified by PCR using primers that added FseI and AscI restrictionsties at the 5′ and 3′ termini respectively. PCR primers ZC26665 (SEQ IDNO:20) and ZC26666 (SEQ ID NO:21) were used with pINCY template plasmidcontaining the full-length ZCYTO18 cDNA in a PCR reaction as follows:one cycle at 95° C. for 5 minutes; followed by 18 cycles at 95° C. for0.5 min., 58° C. for 0.5 min., and 72° C. for 0.5 min.; followed by 72°C. for 7 min.; followed by a 4° C. soak. The PCR reaction product wasloaded onto a 1.2% (low melt) SeaPlaque GTG (FMC, Rockland, Me.) gel inTAE buffer. The ZCYTO18 PCR product was excised from the gel and the gelslice melted at 70μ° C., extracted twice with an equal volume of Trisbuffered phenol, and EtOH precipitated.

The 540 by ZCYTO18 PCR product was digested with FseI and AscI enzymes.The cDNA was isolated on a 1% low melt SeaPlaque GTG™ (FMC, Rockland,Me.) gel and was then excised from the gel and the gel slice melted at70° C., extracted twice with an equal volume of Tris buffered phenol,and EtOH precipitated. The DNA was resuspended in 10 μl H₂O.

The ZCYTO18 cDNA was cloned into the FseI-AscI sites of a modifiedpAdTrack CMV (He, T-C. et al., PNAS 95:2509-2514, 1998). This constructcontains the GFP marker gene. The CMV promoter driving GFP expressionwas replaced with the SV40 promoter and the SV40 polyadenylation signalwas replaced with the human growth hormone polyadenylation signal. Inaddition, the native polylinker was replaced with FseI, EcoRV, and AscIsites. This modified form of pAdTrack CMV was named pZyTrack. Ligationwas performed using the Fast-Link™ DNA ligation and screening kit(Epicentre Technologies, Madison, Wis.). Clones containing the ZCYTO18insert were identified by standard mini prep analysis. The clonedZCYTO18 cDNA was sequenced to verify no errors were introduced duringPCR. In order to linearize the plasmid, approximately 5 μg of thepZyTrack ZCYTO18 plasmid was digested with PmeI. Approximately 1 μg ofthe linearized plasmid was cotransformed with 200 ng of supercoiledpAdEasy (He et al., supra.) into BJ5183 cells. The co-transformation wasdone using a Bio-Rad Gene Pulser at 2.5 kV, 200 ohms and 25mFa. Theentire co-transformation was plated on 4 LB plates containing 25 μg/mlkanamycin. The smallest colonies were picked and expanded inLB/kanamycin and recombinant adenovirus DNA identified by standard DNAminiprep procedures. Digestion of the recombinant adenovirus DNA withFseI-AscI confirmed the presence of ZCYTO18. The recombinant adenovirusminiprep DNA was transformed into DH10B competent cells and DNA preparedusing a Qiagen maxi prep kit as per kit instructions.

Transfection of 293a Cells with Recombinant DNA

Approximately 5 μg of recombinant adenoviral DNA was digested with PacIenzyme (New England Biolabs) for 3 hours at 37° C. in a reaction volumeof 100 μl containing 20-30 U of PacI. The digested DNA was extractedtwice with an equal volume of phenol/chloroform and precipitated withethanol. The DNA pellet was resuspended in 5 μl distilled water. A T25flask of QBI-293A cells (Quantum Biotechnologies, Inc. Montreal, Qc.Canada), inoculated the day before and grown to 60-70% confluence, weretransfected with the PacI digested DNA. The PacI-digested DNA wasdiluted up to a total volume of 50 μl with sterile HBS (150 mM NaCl, 20mM HEPES). In a separate tube, 25 DOTAP (Boehringer Mannheim, 1 mg/ml)was diluted to a total volume of 100 μl with HBS. The DNA was added tothe DOTAP, mixed gently by pipeting up and down, and left at roomtemperature for 15 minutes. The media was removed from the 293A cellsand washed with 5 ml serum-free MEMalpha (Gibco BRL) containing 1 mMSodium Pyruvate (GibcoBRL), 0.1 mM MEM non-essential amino acids(GibcoBRL) and 25 mM HEPES buffer (GibcoBRL). 5 ml of serum-free MEM wasadded to the 293A cells and held at 37° C. The DNA/lipid mixture wasadded drop-wise to the T25 flask of 293A cells, mixed gently andincubated at 37° C. for 4 hours. After 4 hours the media containing theDNA/lipid mixture was aspirated off and replaced with 5 ml complete MEMcontaining 5% fetal bovine serum. The transfected cells were monitoredfor Green Fluorescent Protein (GFP) expression and formation of foci,i.e., viral plaques.

Seven days after transfection of 293A cells with the recombinantadenoviral DNA, the cells expressed the GFP protein and started to formfoci. These foci are viral “plaques” and the crude viral lysate wascollected by using a cell scraper to collect all of the 293A cells. Thelysate was transferred to a 50 ml conical tube. To release most of thevirus particles from the cells, three freeze/thaw cycles were done in adry ice/ethanol bath and a 37° C. waterbath.

Amplification of Recombinant Adenovirus (rAdV)

The crude lysate was amplified (Primary)(1° amplification) to obtain aworking “stock” of zsig45 rAdV lysate. Ten 10 cm plates of nearlyconfluent (80-90%) 293A cells were set up 20 hours previously, 200 ml ofcrude rAdV lysate added to each 10 cm plate and monitored for 48 to 72hours looking for CPE under the white light microscope and expression ofGFP under the fluorescent microscope. When all of the 293A cells showedCPE (Cytopathic Effect) this 1° stock lysate was collected andfreeze/thaw cycles performed as described under Crude rAdV Lysate.

Secondary)(2° Amplification of zsig46 rAdV was obtained as follows:Twenty 15 cm tissue culture dishes of 293A cells were prepared so thatthe cells were 80-90% confluent. All but 20 mls of 5% MEM media wasremoved and each dish was inoculated with 300-500 ml 1° amplified rAdvlysate. After 48 hours the 293A cells were lysed from virus productionand this lysate was collected into 250 ml polypropylene centrifugebottles and the rAdV purified.

rAdV/cDNA Purification

NP-40 detergent was added to a final concentration of 0.5% to thebottles of crude lysate in order to lyse all cells. Bottles were placedon a rotating platform for 10 min. agitating as fast as possible withoutthe bottles falling over. The debris was pelleted by centrifugation at20,000×G for 15 minutes. The supernatant was transferred to 250 mlpolycarbonate centrifuge bottles and 0.5 volumes of 20% PEG8000/2.5MNaCl solution added. The bottles were shaken overnight on ice. Thebottles were centrifuged at 20,000×G for 15 minutes and supernatantdiscarded into a bleach solution. The white precipitate in two verticallines along the wall of the bottle on either side of the spin mark isthe precipitated virus/PEG. Using a sterile cell scraper, theprecipitate from 2 bottles was resuspended in 2.5 ml PBS. The virussolution was placed in 2 ml microcentrifuge tubes and centrifuged at14,000×G in the microfuge for 10 minutes to remove any additional celldebris. The supernatant from the 2 ml microcentrifuge tubes wastransferred into a 15 ml polypropylene snapcap tube and adjusted to adensity of 1.34 g/ml with cesium chloride (CsCl). The volume of thevirus solution was estimated and 0.55 g/ml of CsCl added. The CsCl wasdissolved and 1 ml of this solution weighed 1.34 g. The solution wastransferred polycarbonate thick-walled centrifuge tubes 3.2 ml (Beckman#362305) and spin at 80,000 rpm (348,000 X G) for 3-4 hours at 25° C. ina Beckman Optima TLX microultracentrifuge with the TLA-100.4 rotor. Thevirus formed a white band. Using wide-bore pipette tips, the virus bandwas collected.

The virus from the gradient has a large amount of CsCl which must beremoved before it can be used on cells. Pharmacia PD-10 columnsprepacked with Sephadex G-25M (Pharmacia) were used to desalt the viruspreparation. The column was equilibrated with 20 ml of PBS. The viruswas loaded and allow it to run into the column. 5 ml of PBS was added tothe column and fractions of 8-10 drops collected. The optical densitiesof 1:50 dilutions of each fraction was determined at 260 nm on aspectrophotometer. A clear absorbance peak was present between fractions7-12. These fractions were pooled and the optical density (OD) of a 1:25dilution determined A formula is used to convert OD into virusconcentration: (OD at 260 nm)(25)(1.1×10¹²)=virions/ml. The OD of a 1:25dilution of the ZCYTO18 rAdV was 0.134, giving a virus concentration of3.7×10¹² virions/ml.

To store the virus, glycerol was added to the purified virus to a finalconcentration of 15%, mixed gently but effectively, and stored inaliquots at −80° C.

Tissue Culture Infectious Dose at 50% CPE (TCID 50) Viral TitrationAssay

A protocol developed by Quantum Biotechnologies, Inc. (Montreal, Qc.Canada) was followed to measure recombinant virus infectivity. Briefly,two 96-well tissue culture plates were seeded with 1×10⁴ 293A cells perwell in MEM containing 2% fetal bovine serum for each recombinant virusto be assayed. After 24 hours 10-fold dilutions of each virus from1×10⁻² to 1×10¹⁴ were made in MEM containing 2% fetal bovine serum. 100μl of each dilution was placed in each of 20 wells. After 5 days at 37°C., wells were read either positive or negative for Cytopathic Effect(CPE) and a value for “Plaque Forming Units/ml” (PFU) is calculated.

TCID₅₀ formulation used was as per Quantum Biotechnologies, Inc., above.The titer (T) is determined from a plate where virus used is dilutedfrom 10⁻² to 10⁻¹⁴, and read 8 days after the infection. At eachdilution a ratio (R) of positive wells for CPE per the total number ofwells is determined.

To Calculate titer of the undiluted virus sample: the factor,“F”=1+d(S−0.5); where “S” is the sum of the ratios (R); and “d” is Log10 of the dilution series, for example, “d” is equal to 1 for a ten-folddilution series. The titer of the undiluted sample isT=10^((1+F))=TCID₅₀/ml. To convert TCID₅₀/ml to pfu/ml, 0.7 issubtracted from the exponent in the calculation for titer (T). TheZCYTO18 adenovirus had a titer of 2.8×10¹¹ pfu/ml.

Example 11 In vivo Affects of ZCYTO18 Polypeptide

Mice (female, C57B1, 8 weeks old; Charles River Labs, Kingston, N.Y.)were divided into three groups. On day 0, parental or ZCYTO18 adenovirus(Example 10) was administered to the first (n=8) and second (n=8)groups, respectively, via the tail vein, with each mouse receiving adose of ˜1×10¹¹ particles in ±0.1 ml volume. The third group (n=8)received no treatment. On days 12, mice were weighed and blood was drawnfrom the mice. Samples were analyzed for complete blood count (CBC) andserum chemistry. Statistically significant elevations in neutrophil andplatelet counts were detected in the blood samples from the ZCYTO18adenovirus administered group relative to the parental adenovirustreated group. Also, at day 12 lymphocyte counts were significantlyreduced from the ZCYTO18 adenovirus administered group relative to theparental adenovirus treated group, and they rebounded to normal levelsby day 21. In addition, the ZCYTO18 adenovirus treated mice decreased inbody weight, while parental adenovirus treated mice gained weight. Theelevated platelet and neutrophil count, and the loss of body weight arestill significant as compared to the control group. The liver chemistrytest indicated the increased level of globulin and decreased level ofalbumin concentration, which is consistent with the observation ofinflammatory response induced by TNF-.

The results suggested that ZCYTO18 affects hematopoiesis, i.e., bloodcell formation in vivo. As such, ZCYTO18 could have biologicalactivities affecting different blood precursors, progenitors or stemcells, and a resulting increase or decrease of certain differentiatedblood cells in a specific lineage. For instance, ZCYTO18 appears toreduce lymphocytes, which is likely due to inhibition of the committedprogenitor cells that give rise to lymphoid cells. This finding agreeswith the inhibitory effects of ZCYTO18 on the proliferation and/orgrowth of myeloid stem cells (Example 23), supporting the notion thatZCYTO18 could play a role in anemia, infection, inflammation, and/orimmune diseases by influencing blood cells involved in these process.Antagonists against ZCYTO18, such as antibodies or a soluble receptorantagonist could be used as therapeutic reagents in these diseases. Itis also possible that ZCYTO18 directly affects the release and survivalof platelets in peripheral blood or other vascularized tissues such asliver. That is, besides working through a hematopoisis loop(differentiation, proliferation of stem cells), zcyto18 might directlyaffect the release, stablization or depletion of platelets andneutrophils in peripheral blood or some target tissue and organs.ZCYTO18 also affects the number of granulocytes in the peripheral blood.Extramedullary sites of hematopoiesis (e.g. liver) are also targets forZCYTO18 action.

Moreover, these experiments using ZCYTO18 adenovirus in mice suggestthat ZCYTO18 over-expression increases the level of neutrophils andplatelets in vivo. Although increasing neutrophils and platelets isdesirable in certain therapeutic applications discussed herein, chronicelevation or increased reactivity of these cells could play a role incardiovascular disease. Antagonists against ZCYTO18, such as antibodiesor its soluble receptor, could be used as therapeutic reagents in thesediseases. Although this may appear contradictory to the finding seen inK562 cells (Example 12), it is not uncommon to observe diverseactivities of a particular protein in vitro versus in vivo. It isconceivable that there are other factors (such as cytokines and modifiergenes) involved in the responses to ZCYTO18 in the whole animal system.Nevertheless, these data strongly support the involvement of ZCYTO18 inhematopoiesis. Thus, ZCYTO18 and its receptors are suitablereagents/targets for the diagnosis and treatment in variety ofdisorders, such as inflammation, immune disorders, infection, anemia,hematopoietic and other cancers, and the like.

Example 12 The ZCYTO18 Polypeptide Inhibits the Growth of K-562 Cells inA Cytotoxicity Assay

The K-562 cell line (CRL-243, ATCC) has attained widespread use as ahighly sensitive in vitro target for cytotoxicity assays. K-562 blastsare multipotential, hematopoietic malignant cells that spontaneouslydifferentiate into recognizable progenitors of the erythrocytic,granulocytic and monocytic series (Lozzio, B B et al., Proc. Soc. Exp.Biol. Med. 166: 546-550, 1981).

K562 cells were plated at 5,000 cells/well in 96-well tissue cultureclusters (Costar) in DMEM phenol-free growth medium (Life Technologies)supplemented with pyruvate and 10% serum (HyClone). Next day, humanrecombinant ZCYTO18 (Example 19), BSA control or retinoic acid (known tobe cytotoxic to K562 cells) were added. Seventy-two hours later, thevital stain MTT (Sigma, St Louis, Mo.), a widely used indicator ofmitochondrial activity and cell growth, was added to the cells at afinal concentration of 0.5 mg/ml. MTT is converted to a purple formazanderivative by mitochondrial dehydrogenases. Four hours later, convertedMTT was solubilized by adding an equal volume of acidic isopropanol(0.04N HCl in absolute isopropanol) to the wells. Absorbance wasmeasured at 570 nm, with background subtraction at 650 nm. In thisexperimental setting, absorbance reflects cell viability. Results shownin Table 7 are expressed as % cytotoxicity.

TABLE 7 Agent Concentration % Cytotoxicity BSA Control  1 ug/ml 1.3Retinoic acid 100 uM 62 ZCYTO18 100 ng/ml 16.2 ZCYTO18 300 ng/ml 32

The results indicate that ZCYTO18 may affect myeloid stem cells. Myeloidstem cells are daughter cells of the universal blood stem cells. Theyare progenitors of erythrocytes, monocytes (or migrated macrophages),neutrophil, basophil, and eosinophils. Since K-562 blasts differentiateinto progenitors of the erythrocytic, granulocytic and monocytic series,they are considered a model for myeloid stem cells. Thus, the resultsdemonstrate that ZCYTO18 has an inhibitory activity on the proliferationand/or growth of a promyelocytic tumor cell line. Thus ZCYTO18 couldplay a role in anemia, infection, inflammation, and/or immune diseases.In addition, an antagonist against ZCYTO18, such as antibodies or asoluble receptor antagonist, could be used to block ZCYTO18's activityon myeloid stem cells, or as therapeutic reagents in diseases such asanemia, infection, inflammation, and/or immune diseases. However, asZCYTO18 exhibits cytotoxicity on tumor cells, it can be used directly orin combination with other cytokines as an anti-tumor agent as ananti-tumor agent.

Example 13 Human Zcytor16 Tissue Distribution in Tissue Panels UsingNorthern Blot and PCR

A. Human Zcytor16 Tissue Distribution Using Northern Blot and Dot Blot

Commonly owned, human zcytor16 (SEQ ID NO:32, and SEQ ID NO:33) (PCTInternational Application No. PCT/US00/32703, filed on Dec. 1, 2000) isa naturally-expressed soluble receptor antagonist of ZCYTO18. Northernblot analysis was performed using Human Multiple Tissue Northern BlotsI, II, III (Clontech) and an in house generated U-937 northern blot.U-937 is a human promonocytic blast cell line. The cDNA probe wasgenerated using oligos ZC25,963 (SEQ ID NO:24) and ZC28,354 (SEQ IDNO:25). The PCR conditions were as follows: 94° for 1 minute; 30 cyclesof 94°, 15 seconds; 60°, 30 seconds; 72°, 30 seconds and a finalextension for 5 minutes at 72°. The 364 bp product was gel purified bygel electrophoresis on a 1% TBE gel and the band was excised with arazor blade. The cDNA was extracted from the agarose using the QlAquickGel Extraction Kit (Qiagen). 94 ng of this fragment was radioactivelylabeled with ³²P-dCTP using Rediprime II (Amersham), a random primelabeling system, according to the manufacturer's specifications.Unincorporated radioactivity was removed using a Nuc-Trap column(Stratagene) according to manufacturer's instructions. Blots wereprehybridized at 65° for 3 hours in ExpressHyb (Clontech) solution.Blots were hybridized overnight at 65° in Expresshyb solution containing1.0×10⁶ cpm/ml of labeled probe, 0.1 mg/ml of salmon sperm DNA and 0.5μg/ml of human cot-1 DNA. Blots were washed in 2×SSC, 0.1% SDS at roomtemperature with several solution changes then washed in 0.1×SSC. 0.1%SDS at 55° for 30 minutes twice. Transcripts of approximately 1.6 kb and3.0 kb size were detected in spleen and placenta, but not other tissuesexamined. The same sized transcripts plus an additional approximate 1.2kb transcript was detected in U-937 cell line.

B. Tissue Distribution in Tissue cDNA Panels Using PCR

A panel of cDNAs from human tissues was screened for zcytor16 expressionusing PCR. The panel was made in-house and contained 94 marathon cDNAand cDNA samples from various normal and cancerous human tissues andcell lines are shown in Table 8, below. The cDNAs came from in-houselibraries or marathon cDNAs from in-house RNA preps, Clontech RNA, orInvitrogen RNA. The marathon cDNAs were made using the marathon-Ready™kit (Clontech, Palo Alto, Calif.) and QC tested with clathrin primersZC21195 (SEQ ID NO:26) and ZC21196 (SEQ ID NO:27) and then diluted basedon the intensity of the clathrin band. To assure quality of the panelsamples, three tests for quality control (QC) were run: (1) To assessthe RNA quality used for the libraries, the in-house cDNAs were testedfor average insert size by PCR with vector oligos that were specific forthe vector sequences for an individual cDNA library; (2) Standardizationof the concentration of the cDNA in panel samples was achieved usingstandard PCR methods to amplify full length alpha tubulin or G3PDH cDNAusing a 5′ vector oligo ZC14,063 (SEQ ID NO:28) and 3′ alpha tubulinspecific oligo primer ZC17,574 (SEQ ID NO:29) or 3′ G3PDH specific oligoprimer ZC17,600 (SEQ ID NO:30); and (3) a sample was sent to sequencingto check for possible ribosomal or mitochondrial DNA contamination. Thepanel was set up in a 96-well format that included a human genomic DNA(Clontech, Palo Alto, Calif.) positive control sample. Each wellcontained approximately 0.2-100 pg/μl of cDNA. The PCR reactions wereset up using oligos ZC25,963 (SEQ ID NO:24) and ZC27,659 (SEQ ID NO:25),Advantage 2 DNA Polymerase Mix (Clontech) and Rediload dye (ResearchGenetics, Inc., Huntsville, Ala.). The amplification was carried out asfollow: 1 cycle at 94° C. for 2 minutes, 30 cycles of 94° C. for 20seconds, 58° C. for 30 seconds and 72° C. forl minute, followed by 1cycle at 72° C. for 5 minutes. About 10 μl of the PCR reaction productwas subjected to standard Agarose gel electrophoresis using a 2% agarosegel. The correct predicted DNA fragment size was not observed in anytissue or cell line. Subsequent experiments showing expression ofzcytor16 indicated that the negative results from this panel were likelydue to the primers used.

TABLE 8 Tissue/Cell line #samples Tissue/Cell line #samples Adrenalgland 1 Bone marrow 3 Bladder 1 Fetal brain 3 Bone Marrow 1 Islet 2Brain 1 Prostate 3 Cervix 1 RPMI #1788 2 (ATCC # CCL-156) Colon 1 Testis4 Fetal brain 1 Thyroid 2 Fetal heart 1 WI38 (ATCC # CCL-75 2 Fetalkidney 1 ARIP (ATCC # CRL-1674 - rat) 1 Fetal liver 1 HaCat - humankeratinocytes 1 Fetal lung 1 HPV (ATCC # CRL-2221) 1 Fetal muscle 1Adrenal gland 1 Fetal skin 1 Prostate SM 2 Heart 2 CD3+ selected PBMC's1 Ionomycin + PMA stimulated K562 1 HPVS (ATCC # CRL-2221) - 1 (ATCC #selected CCL-243) Kidney 1 Heart 1 Liver 1 Pituitary 1 Lung 1 Placenta 2Lymph node 1 Salivary gland 1 Melanoma 1 HL60 (ATCC # CCL-240) 3Pancreas 1 Platelet 1 Pituitary 1 HBL-100 1 Placenta 1 Renal mesangial 1Prostate 1 T-cell 1 Rectum 1 Neutrophil 1 Salivary Gland 1 MPC 1Skeletal muscle 1 Hut-102 (ATCC # TIB-162) 1 Small intestine 1Endothelial 1 Spinal cord 1 HepG2 (ATCC # HB-8065) 1 Spleen 1 Fibroblast1 Stomach 1 E. Histo 1 Testis 2 Thymus 1 Thyroid 1 Trachea 1 Uterus 1Esophagus 1 tumor Gastric tumor 1 Kidney tumor 1 Liver tumor 1 Lungtumor 1 Ovarian tumor 1 Rectal tumor 1 Uterus tumor 1

An additional panel of cDNAs from human tissues was screened forzcytor16 expression using PCR. This panel was made in-house andcontained 77 marathon cDNA and cDNA samples from various normal andcancerous human tissues and cell lines are shown in Table 9, below.Aside from the PCR reaction, the assay was carried out as per above. ThePCR reactions were set up using oligos ZC25,963 (SEQ ID NO:24) andZC25,964 (SEQ ID NO:31), Advantage 2 DNA Polymerase Mix (Clontech) andRediload dye (Research Genetics, Inc., Huntsville, Ala.). Theamplification was carried out as follow: 1 cycle at 94° C. for 1 minute,38 cycles of 94° C. for 10 seconds, 60° C. for 30 seconds and 72° C. for30 seconds, followed by 1 cycle at 72° C. for 5 minutes. The correctpredicted DNA fragment size was observed in bone marrow, fetal heart,fetal kidney, fetal muscle, fetal skin, heart, mammary gland, placenta,salivary gland, skeletal muscle, small intestine, spinal cord, spleen,kidney, fetal brain, esophageal tumor, uterine tumor, stomach tumor,ovarian tumor, rectal tumor, lung tumor and RPMI-1788 (a B-lymphocytecell line). Zcytor16 expression was not observed in the other tissuesand cell lines tested in this panel. The expression pattern of zcytor16shows expression in certain tissue-specific tumors especially, e.g.,ovarian cancer, stomach cancer, uterine cancer, rectal cancer, lungcancer and esophageal cancer, where zcytor16 is not expressed in normaltissue, but is expressed in the tumor tissue. One of skill in the artwould recognize that the natural ligand, CYTO18, and receptor bindingfragments of ZCYTO18 of the present invention can be used as adiagnostic to detect cancer, or cancer tissue in a biopsy, tissue, orhistologic sample, particularly e.g., ovarian cancer, stomach cancer,uterine cancer, rectal cancer, lung cancer and esophageal cancer tissue.Such diagnostic uses for the molecules of the present invention areknown in the art and described herein.

In addition, because the expression pattern of zcytor16, one ofZCYTO18's receptors, shows expression in certain specific tissues aswell as tissue-specific tumors, binding partners including the naturalligand, ZCYTO18, can also be used as a diagnostic to detect specifictissues (normal or abnormal), cancer, or cancer tissue in a biopsy,tissue, or histologic sample, where ZCYTO18 receptors are expressed, andparticularly e.g., ovarian cancer, stomach cancer, uterine cancer,rectal cancer, lung cancer and esophageal cancer tissue. ZCYTO18 canalso be used to target other tissues wherein its receptors, e.g.,zcytor16 and zcytor11 (Commonly owned U.S. Pat. No. 5,965,704) areexpressed. Moreover, such binding partners could be conjugated tochemotherapeutic agents, toxic moieties and the like to target therapyto the site of a tumor or diseased tissue. Such diagnostic and targetedtherapy uses are known in the art and described herein.

A commercial 1st strand cDNA panel (Human Blood Fractions MTC Panel,Clontech, Palo Alto, Calif.) was also assayed as above. The panelcontained the following samples: mononuclear cells, activatedmononuclear cells, resting CD4+ cells, activated CD4+ cells, restingCD8+ cells, activated CD8+ cells, resting CD14+ cells, resting CD19+cells and activated CD19+ cells. Activated CD4+ cells and activatedCD19+ cells showed zcytor16 expression, whereas the other cells tested,including resting CD4+ cells and resting CD19+ cells, did not.

TABLE 9 Tissue #samples Tissue #samples adrenal gland 1 bladder 1 bonemarrow 3 brain 2 cervix 1 colon 1 fetal brain 3 fetal heart 2 fetalkidney 1 fetal liver 2 fetal lung 1 fetal skin 1 heart 2 fetal muscle 1kidney 2 liver 1 lung 1 lymph node 1 mammary gland 1 melanoma 1 ovary 1pancreas 1 pituitary 2 placenta 3 prostate 3 rectum 1 salivary gland 2skeletal muscle 1 small intestine 1 spinal cord 2 spleen 1 uterus 1stomach 1 adipocyte library 1 testis 5 islet 1 thymus 1 prostate SMC 1thyroid 2 RPMI 1788 1 trachea 1 WI38 1 esophageal tumor 1 lung tumor 1liver tumor 1 ovarian tumor 1 rectal tumor 1 stomach tumor 1 uterinetumor 2 CD3+ library 1 HaCAT library 1 HPV library 1 HPVS library 1 MG63library 1 K562 1C. Tissue Distribution in Human Tissue and Cell Line RNA Panels UsingRT-PCR

A panel of RNAs from human cell lines was screened for zcytor16expression using RT-PCR. The panels were made in house and contained 84RNAs from various normal and cancerous human tissues and cell lines asshown in Tables 10-13 below. The RNAs were made from in house orpurchased tissues and cell lines using the RNAeasy Midi or Mini Kit(Qiagen, Valencia, Calif.). The panel was set up in a 96-well formatwith 100 ngs of RNA per sample. The RT-PCR reactions were set up usingoligos ZC25,963 (SEQ ID NO:24) and ZC25,964 (SEQ ID NO:31), Rediload dyeand SUPERSCRIPT One Step RT-PCR System (Life Technologies, Gaithersburg,Md.). The amplification was carried out as follows: one cycle at 55° for30 minutes followed by 40 cycles of 94°, 15 seconds; 59°, 30 seconds;72°, 30 seconds; then ended with a final extension at 72° for 5 minutes.8 to 10 μls of the PCR reaction product was subjected to standardAgarose gel electrophoresis using a 4% agarose gel. The correctpredicted cDNA fragment size of 184 bps was observed in cell linesU-937, HL-60, ARPE-19, HaCat#1, HaCat#2, HaCat#3, and HaCat#4; bladder,cancerous breast, normal breast adjacent to a cancer, bronchus, colon,ulcerative colitis colon, duodenum, endometrium, esophagus,gastro-esophageal, heart left ventricle, heart ventricle, ileum, kidney,lung, lymph node, lymphoma, mammary adenoma, mammary gland, cancerousovary, pancreas, parotid and skin, spleen lymphoma and small bowel.Zcytor16 expression was not observed in the other tissues and cell linestested in this panel.

Zcytor16 is detectably expressed by PCR in normal tissues: such as, thedigestive system, e.g., esophagus, gastro-esophageal, pancreas,duodenum, ileum, colon, small bowel; the female reproductive system,e.g., mammary gland, endometrium, breast (adjacent to canceroustissues); and others systems, e.g., lymph nodes, skin, parotid, bladder,bronchus, heart ventricles, and kidney. Moreover, Zcytor16 is detectablyexpressed by PCR in several human tumors: such as tumors associated withfemale reproductive tissues e.g., mammary adenoma, ovary cancer, uterinecancer, other breast cancers; and other tissues such as lymphoma,stomach tumor, and lung tumor. The expression of zcytor16 is found innormal tissues of female reproductive organs, and in some tumorsassociated with these organs. As such, a ligand for zcytor16, such asZCYTO18, or a receptor-binding fragment thereof, can serve as a markerfor these tumors wherein the zcytor16 may be over-expressed. Severalcancers positive for zcytor16 are associated with ectodermal/epithelialorigin (mammary adenoma, and other breast cancers). Hence, ligand forzcytor16, such as ZCYTO18, or a receptor-binding fragment thereof, canserve as a marker for epithelial tissue, such as epithelial tissues inthe digestive system and female reproductive organs (e.g., endometrialtissue, columnar epithelium), as well as cancers involving epithelialtissues. Moreover, in a preferred embodiment, ZCYTO18, or areceptor-binding fragment thereof, can serve as a marker for certaintissue-specific tumors especially, e.g., ovarian cancer, stomach cancer,uterine cancer, rectal cancer, lung cancer and esophageal cancer, whereit's receptor zcytor16 is not expressed in normal tissue, but isexpressed in the tumor tissue. Use of polynucleotides, polypeptides, andantibodies of the present invention for diagnostic purposes are known inthe art, and disclosed herein.

TABLE 10 Tissue #samples Tissue #samples adrenal gland 6 duodenum 1bladder 3 endometrium 5 brain 2 cancerous endometrium 1 brain meningioma1 gastric cancer 1 breast 1 esophagus 7 cancerous breast 4gastro-esophageal 1 normal breast 5 heart aorta 1 adjacent to cancerbronchus 3 heart left ventricle 4 colon 15 heart right ventricle 2cancerous colon 1 heart ventricle 1 normal colon 1 ileum 3 adjacent tocancer ulcerative colitis colon 1 kidney 15 cancerous kidney 1

TABLE 11 Tissue/Cell Line #samples Tissue/Cell Line #samples 293 1HBL-100 1 C32 1 Hs-294T 1 HaCat#1 1 Molt4 1 HaCat#2 1 RPMI 1 HaCat#3 1U-937 1 HaCat#4 1 A-375 1 WI-38 1 HCT-15 1 WI-38 + 2 um ionomycin #1 1HT-29 1 WI-38 + 2 um ionomycin #2 1 MRC-5 1 WI-38 + 5 um ionomycin#1 1RPT-1 1 WI-38 + 5 um ionomycin#2 1 RPT-2 1 Caco-2, 1 WM-115 1 Caco-2,differentiated 1 A-431 1 DLD-1 1 WERI-Rb-1 1 HRE 1 HEL-92.1.7 1 HRCE 1HuH-7 1 MCF7 1 MV-4-11 1 PC-3 1 U-138 1 TF-1 1 CCRF-CEM 1 5637 1 Y-79 1143B 1 A-549 1 ME-180 1 EL-4 1 prostate epithelia 1 HeLa 229 1 U-2 OS 1HUT 78 1 T-47D 1 NCI-H69 1 Mg-63 1 SaOS2 1 Raji 1 USMC 1 U-373 MG 1UASMC 2 A-172 1 AoSMC 1 CRL-1964 1 UtSMC 1 CRL-1964 + butryic acid 1HepG2 1 HUVEC 1 HepG2-IL6 1 SK-Hep-1 1 NHEK#1 1 SK-Lu-1 1 NHEK#2 1Sk-MEL-2 1 NHEK#3 1 K562 1 NHEK#4 1 BeWo 1 ARPE-19 1 FHS74.Int 1 G-361 1HL-60 1 HISM 1 Malme 3M 1 3AsubE 1 FHC 1 INT407 1 HREC 1

TABLE 12 Tissue #samples Tissue #samples liver 10 lung 13 lymph node 1cancerous lung 2 lymphoma 4 normal lung adjacent 1 to cancer mammaryadenoma 1 muscle 3 mammary gland 3 neuroblastoma 1 melinorioma 1 omentum2 osteogenic sarcoma 2 ovary 6 pancreas 4 cancerous ovary 2 skin 5parotid 7 sarcoma 2 salivary gland 4

TABLE 13 Tissue #samples Tissue #samples small bowel 10 uterus 11 spleen3 uterine cancer 1 spleen lymphoma 1 thyroid 9 stomach 13 stomach cancer1

Example 14 Human Zcytor11 Tissue Distribution in Tissue Panels UsingNorthern Blot and PCR

A. Human Zcytor11 Tissue Distribution in Tissue Panels Using PCR

A panel of cDNAs from human tissues was screened for zcytor11 expressionusing PCR. Commonly owned, human zcytor11 (SEQ ID NO:18, and SEQ IDNO:19) (U.S. Pat. No. 5,965,704) is a receptor for ZCYTO18. The panelwas made in-house and contained 94 marathon cDNA and cDNA samples fromvarious normal and cancerous human tissues and cell lines are shown inTable 9 above. Aside from the PCR reaction, the method used was as shownin Example 13. The PCR reactions were set up using oligos ZC14,666 (SEQID NO:22) and ZC14,742 (SEQ ID NO:23), Advantage 2 cDNA polymerase mix(Clontech, Palo Alto, Calif.), and Rediload dye (Research Genetics,Inc., Huntsville, Ala.). The amplification was carried out as follows: 1cycle at 94° C. for 2 minutes, 40 cycles of 94° C. for 15 seconds, 51°C. for 30 seconds and 72° C. for 30 seconds, followed by 1 cycle at 72°C. for 7 minutes. The correct predicted DNA fragment size was observedin bladder, brain, cervix, colon, fetal brain, fetal heart, fetalkidney, fetal liver, fetal lung, fetal skin, heart, kidney, liver, lung,melanoma, ovary, pancreas, placenta, prostate, rectum, salivary gland,small intestine, testis, thymus, trachea, spinal cord, thyroid, lungtumor, ovarian tumor, rectal tumor, and stomach tumor. Zcytor11expression was not observed in the other tissues and cell lines testedin this panel.

A commercial 1st strand cDNA panel (Human Blood Fractions MTC Panel,Clontech, Palo Alto, Calif.) was also assayed as above. The panelcontained the following samples: mononuclear cells, activatedmononuclear cells, resting CD4+ cells, activated CD4+ cells, restingCD8+ cells, activated CD8+ cells, resting CD14+ cells, resting CD19+cells and activated CD19+ cells. All samples except activated CD8+ andActivated CD19+ showed expression of zcytor11.

B. Tissue Distribution of Zcytor11 in Human Cell Line and Tissue PanelsUsing RT-PCR

A panel of RNAs from human cell lines was screened for zcytor11expression using RT-PCR. The panels were made in house and contained 84RNAs from various normal and cancerous human tissues and cell lines asshown in Tables 10-13 above. The RNAs were made from in house orpurchased tissues and cell lines using the RNAeasy Midi or Mini Kit(Qiagen, Valencia, Calif.). The panel was set up in a 96-well formatwith 100 ngs of RNA per sample. The RT-PCR reactions were set up usingoligos ZC14,666 (SEQ ID NO:22) and ZC14,742 (SEQ ID NO:23), Rediload dyeand SUPERSCRIPT One Step RT-PCR System(Life Technologies, Gaithersburg,Md.). The amplification was carried out as follows: one cycle at 50° for30 minutes followed by 45 cycles of 94°, 15 seconds; 52°, 30 seconds;72°, 30 seconds; then ended with a final extension at 72° for 7 minutes.8 to 10 uls of the PCR reaction product was subjected to standardAgarose gel electrophoresis using a 4% agarose gel. The correctpredicted cDNA fragment size was observed in adrenal gland, bladder,breast, bronchus, normal colon, colon cancer, duodenum, endometrium,esophagus, gastic cancer, gastro-esophageal cancer, heart ventricle,ileum, normal kidney, kidney cancer, liver, lung, lymph node, pancreas,parotid, skin, small bowel, stomach, thyroid, and uterus. Cell linesshowing expression of zcytor11 were A-431, differentiated CaCO2, DLD-1,HBL-100, HCT-15, HepG2, HepG2+IL6, HuH7, and NHEK #1-4. Zcytor11expression was not observed in the other tissues and cell lines testedin this panel.

In addition, because the expression pattern of zcytor11, one ofZCYTO18's receptors, shows expression in certain specific tissues,binding partners including the natural ligand, ZCYTO18, can also be usedas a diagnostic to detect specific tissues (normal or abnormal), cancer,or cancer tissue in a biopsy, tissue, or histologic sample, particularlyin tissues where ZCYTO18 receptors are expressed. ZCYTO18 can also beused to target other tissues wherein its receptors, e.g., zcytor16 andzcytor11 are expressed. Moreover, such binding partners could beconjugated to chemotherapeutic agents, toxic moieties and the like totarget therapy to the site of a tumor or diseased tissue. Suchdiagnostic and targeted therapy uses are known in the art and describedherein.

The expression patterns of zcytor11 (above) and zcytor16 (Example 13,and Example 15) indicated target tissues and cell types for the actionof ZCYTO18, and hence ZCYTO18 antagonists. The zcytor11 expressiongenerally overlapped with zcytor16 expression in three physiologicsystems: digestive system, female reproductive system, and immunesystem. Moreover, the expression pattern of the receptor (zcytor11)indicated that a ZCYTO18 antagonist such as zcytor16 would havetherapeutic application for human disease in at least two areas:inflammation (e.g., IBD, Chron's disease, pancreatitis) and cancer(e.g., ovary, colon). That is, the polynucleotides, polypeptides andantibodies of the present invention can be used to antagonize theinflammatory, and other cytokine-induced effects of ZCYTO18 interactionwith the cells expressing the zcytor11 receptor.

Moreover, the expression of zcytor11 appeared to be downregulated orabsent in an ulcerative colitis tissue, HepG2 liver cell line induced byIL-6, activated CD8+ T-cells and CD19+ B-cells. However, zcytor16appeared to be upregulated in activated CD19+ B-cells (Example 12),while zcytor11 is downregulated in activated CD19+ cells, as compared tothe resting CD19+ cells (above). The expression of zcytor11 and zcytor16has a reciprocal correlation in this case. These RT-PCR experimentsdemonstrate that CD19+ peripheral blood cells, B lymphocytes, expressreceptors for ZCYTO18, namely zcytor11 and zcytor16. Furthermore B cellsdisplay regulated expression of zcytor11 and zcytor16. B-lymphocytesactivated with mitogens decrease expression of zcytor11 and increaseexpression of zcytor16. This represents feedback inhibition that wouldserve to dampen the activity of ZCYTO18 on B cells and other cells aswell. Soluble zcytor16 would act as an antagonist to neutralize theeffects of ZCYTO18 on B cells. This would be beneficial in diseaseswhere B cells are the key players: Autoimmune diseases includingsystemic lupus erythmatosus (SLE), myasthenia gravis, immune complexdisease, and B-cell cancers that are exacerbated by ZCYTO18. Alsoautoimmune diseases where B cells contribute to the disease pathologywould be targets for zcytor16 therapy: Multiple sclerosis, inflammatorybowel disease (IBD) and rheumatoid arthritis are examples. Zcytor16therapy would be beneficial to dampen or inhibit B cells producing IgEin atopic diseases including asthma, allergy and atopic dermatitis wherethe production of IgE contributes to the pathogenesis of disease.

B cell malignancies may exhibit a loss of the “feedback inhibition”described above. Administration of zcytor16 would restore control ofZCYTO18 signaling and inhibit B cell tumor growth. The administration ofzcytor16 following surgical resection or chemotherapy may be useful totreat minimal residual disease in patients with B cell malignancies. Theloss of regulation may lead to sustained or increased expression ofzcytor11. Thus creating a target for therapeutic monoclonal antibodiestargeting zcytor11.

Example 15 Identification of Cells Expressing Zcytor16 Using In SituHybridization

Specific human tissues were isolated and screened for zcytor16expression by in situ hybridization. Various human tissues prepared,sectioned and subjected to in situ hybridization included cartilage,colon, appendix, intestine, fetal liver, lung, lymph node, lymphoma,ovary, pancreas, placenta, prostate, skin, spleen, and thymus. Thetissues were fixed in 10% buffered formalin and blocked in paraffinusing standard techniques. Tissues were sectioned at 4 to 8 microns.Tissues were prepared using a standard protocol (“Development ofnon-isotopic in situ hybridization” at The Laboratory of ExperimentalPathology (LEP), NIEHS, Research Triangle Park, NC). Briefly, tissuesections were deparaffinized with HistoClear (National Diagnostics,Atlanta, GA) and then dehydrated with ethanol. Next they were digestedwith Proteinase K (50 μg/ml) (Boehringer Diagnostics, Indianapolis, IN)at 37° C. for 2 to 7 minutes. This step was followed by acetylation andre-hydration of the tissues.

One in situ probe was designed against the human zcytor16 sequence(nucleotide 1-693 of SEQ ID NO:32), and isolated from a plasmidcontaining SEQ ID NO:32 using standard methods. T3 RNA polymerase wasused to generate an antisense probe. The probe was labeled withdigoxigenin (Boehringer) using an In Vitro transcription System(Promega, Madison, Wis.) as per manufacturer's instruction.

In situ hybridization was performed with a digoxigenin-labeled zcytor16probe (above). The probe was added to the slides at a concentration of 1to 5 pmol/ml for 12 to 16 hours at 62.5° C. Slides were subsequentlywashed in 2×SSC and 0.1×SSC at 55° C. The signals were amplified usingtyramide signal amplification (TSA) (TSA, in situ indirect kit; NEN) andvisualized with Vector Red substrate kit (Vector Lab) as permanufacturer's instructions. The slides were then counter-stained withhematoxylin (Vector Laboratories, Burlingame, Calif.).

Signals were observed in several tissues tested: The lymph node, plasmacells and other mononuclear cells in peripheral tissues were stronglypositive. Most cells in the lymphatic nodule were negative. In lymphomasamples, positive signals were seen in the mitotic and multinuclearcells. In spleen, positive signals were seen in scattered mononuclearcells at the periphery of follicles were positive. In thymus, positivesignals were seen in scattered mononuclear cells in both cortex andmedulla were positive. In fetal liver, a strong signal was observed in amixed population of mononuclear cells in sinusoid spaces. A subset ofhepatocytes might also have been positive. In the inflamed appendix,mononuclear cells in peyer's patch and infiltration sites were positive.In intestine, some plasma cells and ganglia nerve cells were positive.In normal lung, zcytor16 was expressed in alveolar epithelium andmononuclear cells in interstitial tissue and circulation. In the lungcarcinoma tissue, a strong signal was observed in mostly plasma cellsand some other mononuclear cells in peripheral of lymphatic aggregates.In ovary carcinoma, epithelium cells were strongly positive. Someinterstitial cells, most likely the mononuclear cells, were alsopositive. There was no signal observed in the normal ovary. In bothnormal and pancreatitis pancreas samples, acinar cells and somemononuclear cells in the mesentery were positive. In the early term (8weeks) placenta, signal was observed in trophoblasts. In skin, somemononuclear cells in the inflamed infiltrates in the superficial dermiswere positive. Keratinocytes were also weakly positive. In prostatecarcinoma, scatted mononuclear cells in interstitial tissues werepositive. In articular cartilage, chondrocytes were positive. Othertissues tested including normal ovary and a colon adenocarcinoma werenegative.

In summary, the in situ data was consistent with expression datadescribed above for the zcytor16. Zcytor16 expression was observedpredominately in mononuclear cells, and a subset of epithelium was alsopositive. These results confirmed the presence of zcytor16 expression inimmune cells and point toward a role in inflammation, autoimmunedisease, or other immune function, for example, in bindingpro-inflammatory cytokines, including but not limited to ZCYTO18.Moreover, detection of zcytor16 expression can be used for example as anmarker for mononuclear cells in histologic samples.

Zcytor16 is expressed in mononuclear cells, including normal tissues(lymph nodes, spleen, thymus, pancreas and fetal liver, lung), andabnormal tissues (inflamed appendix, lung carcinoma, ovary carcinoma,pancreatitis, inflamed skin, and prostate carcinoma). It is notable thatplasma cells in the lymph node, intestine, and lung carcinoma arepositive for zcytor16. Plasma cells are immunologically activatedlymphocytes responsible for antibody synthesis. In addition, ZCYTO18, isexpressed in activated T cells. In addition, the expression of zcytor16is detected only in activated (but not in resting) CD4+ and CD19+ cells(Example 13). Thus, zcytor16 can be used as a marker for or as a targetin isolating certain lymphocytes, such as mononuclear leucocytes andlimited type of activated leucocytes, such as activated CD4+ and CD19+.

Furthermore, the presence of zcytor16 expression in activated immunecells such as activated CD4+ and CD19+ cells showed that zcytor16 may beinvolved in the body's immune defensive reactions against foreigninvaders: such as microorganisms and cell debris, and could play a rolein immune responses during inflammation and cancer formation.

Moreover, as discussed herein, epithelium form several tissues waspositive for zcytor16 expression, such as hepatocytes (endoderm-derivedepithelia), lung alveolar epithelium (endoderm-derived epithelia), andovary carcinoma epithelium (mesoderm-derived epithelium). The epitheliumexpression of zcytor16 could be altered in inflammatory responses and/orcancerous states in liver and lung. Thus, ligand for zcytor16, such asZCYTO18, or a receptor-binding fragment thereof, could be used as markerto monitor changes in these tissues as a result of inflammation orcancer. Moreover, analysis of zcytor16 in situ expression showed thatnormal ovary epithelium is negative for zcytor16 expression, while it isstrongly positive in ovary carcinoma epithelium providing furtherevidence that ZCYTO18 polypeptides, or a receptor-binding fragmentthereof, can be used as a diagnostic marker and/or therapeutic targetfor the diagnosis and treatment of ovarian cancers, and ovary carcinoma,as described herein.

Zcytor16 was also detected in other tissues, such as acinar cells inpancreas (normal and pancreatitis tissues), trophoblasts in placenta(ectoderm-derived), chondrocytes in cartilage (mesoderm-derived), andganglia cells in intestine (ectoderm-derived). As such, zcytor16 may beinvolved in differentiation and/or normal functions of correspondingcells in these organs. As such, potential utilities of zcytor16 includemaintenance of normal metabolism and pregnancy, boneformation/homeostasis, and physiological function of intestine, and thelike.

Example 16 huZCYTO18 Anti-Peptide Antibodies

Polyclonal anti-peptide antibodies were prepared by immunizing twofemale New Zealand white rabbits with the peptide, huZCYTO18-1 (SEQ IDNO:34) or huZCYTO18-2 (SEQ ID NO:35) or huZCYTO18-3 (SEQ ID NO:36). Thepeptides were synthesized using an Applied Biosystems Model 431A peptidesynthesizer (Applied Biosystems, Inc., Foster City, Calif.) according tomanufacturer's instructions. The peptides huZCYTO18-1, huZCYTO18-2, andhuZCYTO18-3 were then conjugated to the carrier proteinmaleimide-activated keyhole limpet hemocyanin (KLH) through cysteineresidues (Pierce, Rockford, Ill.). The rabbits were each given aninitial intraperitoneal (IP) injection of 200 μg of conjugated peptidein Complete Freund's Adjuvant (Pierce, Rockford, Ill.) followed bybooster IP injections of 100 μg conjugated peptide in IncompleteFreund's Adjuvant every three weeks. Seven to ten days after theadministration of the third booster injection, the animals were bled andthe serum was collected. The rabbits were then boosted and bled everythree weeks.

The huZCYTO18 peptide-specific Rabbit seras were characterized by anELISA titer check using 1 μg/ml of the peptide used to make the antibodyas an antibody target. The 2 rabbit seras to the huZCYTO18-1 peptide(SEQ ID NO:34) have titer to their specific peptide at a dilution of 1:5E6 (1:5,000,000).

The huZCYTO18-1 peptide-specific antibodies were affinity purified fromthe rabbit serum using an EPDXY-SEPHAROSE 6B peptide column (PharmaciaLKB) that was prepared using 10 mg of the respective peptides per gramEPDXY-SEPHAROSE 6B, followed by dialysis in PBS overnight.Peptide-specific huZCYTO18 antibodies were characterized by an ELISAtiter check using 1 μg/ml of the appropriate peptide as an antibodytarget. The huZCYTO18-1 peptide-specific antibodies have a lower limitof detection (LLD) of 500 pg/ml by ELISA on its appropriate antibodytarget. The huZCYTO18-1 peptide-specific antibodies recognizefull-length recombinant protein (BV produced) by reducing Western Blotanalysis.

Example 17 Construction of Human ZCYTO18 Transgenic Plasmids

Approximately 10 μg Zytrack vector containing the sequence confirmedhuman ZCYTO18 coding region was digested with FseI and AscI. The vectorwas then ethanol precipitated and the pellet was resuspended in TE. Thereleased 540 by human ZCYTO18 fragment was isolated by running thedigested vector on a 1.2% SeaPlaque gel and excising the fragment. DNAwas purified using the QiaQuick (Qiagen) gel extraction kit.

The human ZCYTO18 fragment was then ligated into pTG12-8, our standardtransgenic vector, which was previously digested with FseI and AscI. ThepTG12-8 plasmid, designed for expression of a gene of interest intransgenic mice, contains an expression cassette flanked by 10 kb ofMT-1 5′ DNA and 7 kb of MT-1 3′ DNA. The expression cassette comprisesthe MT-1 promoter, the rat insulin II intron, a polylinker for theinsertion of the desired clone, and the human growth hormone poly Asequence.

About one microliter of the ligation reaction was electroporated intoDH10B ElectroMax® competent cells (GIBCO BRL, Gaithersburg, Md.)according to manufacturer's direction, plated onto LB plates containing100 μg/ml ampicillin, and incubated overnight at 37° C. Colonies werepicked and grown in LB media containing 100 μg/ml ampicillin. MiniprepDNA was prepared from the picked clones and screened for the humanZCYTO18 insert by restriction digestion with FseI/AscI, and subsequentagarose gel electrophoresis. Maxipreps of the correct pTG12-8 humanZCYTO18 construct were performed.

A SalI fragment containing 5′ and 3′ flanking sequences, the MTpromoter, the rat insulin II intron, human ZCYTO18 cDNA and the humangrowth hormone poly A sequence was prepared and used for microinjectioninto fertilized murine oocytes.

A second transgenic construct was made by subcloning as described above,the FseI/AscI fragment containing the human ZCYTO18 cDNA, into alymphoid-specific transgenic vector pKFO51. The pKFO51 transgenic vectoris derived from p1026X (Iritani, B. M., et al., EMBO J. 16:7019-31,1997) and contains the T cell-specific lck proximal promoter, the B/Tcell-specific immunoglobulin Eμ heavy chain enhancer, a polylinker forthe insertion of the desired clone, and a mutated hGH gene that encodesan inactive growth hormone protein (providing 3′ introns and apolyadenylation signal).

Maxi-prep DNA was digested with NotI, and this fragment, containing thelck proximal promoter, immunoglobulin Eμ enhancer, human ZCYTO18 cDNA,and the mutated hGH gene was prepared to be used for microinjection intofertilized murine oocytes.

Construction of Mouse ZCYTO18 Transgenic Plasmids

Transgenic constructs were also made for mouse ZCYTO18. Oligonucleotideswere designed to generate a PCR fragment containing a consensus Kozaksequence and the exact mouse ZCYTO18 coding region. Theseoligonucleotides were designed with an FseI site at the 5′ end and anAscI site at the 3′ end to facilitate cloning into pKFO51, alymphoid-specific transgenic vector containing the EuLCK promoter todrive expression of ZCYTO18.

PCR reactions were carried out with 200 ng mouse ZCYTO18 template (SEQID NO:37) and oligonucleotides ZC37,125 (SEQ ID NO:39) and ZC37,126 (SEQID NO:40). A PCR reaction was performed using Advantage™ cDNA polymerase(Clontech) under the following conditions: 95° C. for 5 minutes; 15cycles of 95° C. for 60 seconds, 60° C. for 60 seconds, and 72° C. for90 seconds; and 72° C. for 7 minutes. PCR products were separated byagarose gel electrophoresis and purified using a QiaQuick (Qiagen) gelextraction kit. The isolated, 540 bp, DNA fragment was digested withFseI and AscI (Boerhinger-Mannheim), ethanol precipitated and clonedinto pKFO51 as described above. A correct clone of pKFO51 mouse ZCYTO18was verified by sequencing, and a maxiprep of this clone was performedand prepared as above for injection.

Example 18 Baculovirus Expression of zCyto18-CEE

An expression vector, zCyto18-CEE/pZBV32L, was prepared to expresszCyto18-CEE polypeptides in insect cells. zCyto18-CEE/pZBV32L wasdesigned to express a zCyto18 polypeptide with a C-terminal GLU-GLU tag(SEQ ID NO:14). This construct can be used to determine the N-terminalamino acid sequence of zCyto18 after the signal peptide has been cleavedoff.

A. Construction of zCyto18-CEE/pZBV32L

A 561 by zCyto18 fragment containing BamHI and XbaI restriction sites onthe 5′ and 3′ ends, respectively, was generated by PCR amplificationfrom a plasmid containing zCyto18 cDNA using primers ZC28,348 (SEQ IDNO:41) and ZC28,345 (SEQ ID NO:42). The PCR reaction conditions were asfollows: 1 cycle at 94° C. for 5 minutes; 35 cycles of 94° C. for 90seconds, 60° C. for 120 seconds, and 72° C. for 180 seconds; 1 cycle at72° C. for 10 min; followed by 4° C. soak. The fragment was visualizedby gel electrophoresis (1% agarose). The band was excised and thenextracted using a QIAquick™ Gel Extraction Kit (Qiagen, Cat. No. 28704).The cDNA was digested using BamHI and XbaI and then was ligated into thevector pZBV32L. The pZBV32L vector is a modification of the pFastBac1 ™(Life Technologies) expression vector, where the polyhedron promoter hasbeen removed and replaced with the late activating Basic ProteinPromoter, and the coding sequence for the Glu-Glu tag as well as a stopsignal was inserted at the 3′ end of the multiple cloning region.Approximately 68 nanograms of the restriction digested zCyto18 insertand about 100 ng of the corresponding pZBV32L vector were ligatedovernight at 16° C. The ligation mix was diluted 10 fold in water and 1fmol of the diluted ligation mix was transformed into ElectoMAX™ DH12s™cells (Life Technologies, Cat. No. 18312-017) by electroporation at 400Ohms, 2V and 25 μF in a 2 mm gap electroporation cuvette (BTX, Model No.620). The transformed cells were diluted in 450 μl of SOC media (2%Bacto Tryptone, 0.5% Bacto Yeast Extract, 10 ml 1M NaCl, 1.5 mM KCl, 10mM MgCl₂, 10 mM MgSO₄ and 20 mM glucose) and 100 μl of the dilution wereplated onto LB plates containing 100 μg/ml ampicillin. Clones wereanalyzed by PCR and two positive clones were selected to be outgrown andpurified using a QIAprep® Spin Miniprep Kit (Qiagen, Cat. No. 27106).Two μl of each of the positive clones were transformed into 20 μlDH10Bac™ Max Efficiency® competent cells (GIBCO-BRL Cat. No. 10361-012)by heat shock for 45 seconds in a 42° C. heat block. The transformedDH10Bac™ cells were diluted in 980 μl SOC media (2% Bacto Tryptone, 0.5%Bacto Yeast Extract, 10 ml 1M NaCl, 1.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄and 20 mM glucose) and 100 μl were plated onto Luria Agar platescontaining 50 μg/ml kanamycin, 7 μg/ml gentamicin, 10 μg/mltetracycline, 40 μg/mL IPTG and 200 μg/mL Bluo Gal. The plates wereincubated for 48 hours at 37° C. A color selection was used to identifythose cells having transposed viral DNA (referred to as a “bacmid”).Those colonies, which were white in color, were picked for analysis.Colonies were analyzed by PCR and positive colonies (containing desiredbacmid) were selected for outgrowth and purified using a QIAprep® SpinMiniprep Kit (Qiagen, Cat. No. 27106). Clones were screened for thecorrect insert by amplifying DNA using primers to the transposableelement in the bacmid via PCR using primers ZC447 (SEQ ID NO:43) andZC976 (SEQ ID NO:44). The PCR reaction conditions were as follows: 1cycle at 94° C. for 5 minutes; 30 cycles of 94° C. for 60 seconds, 50°C. for 90 seconds, and 72° C. for 180 seconds; 1 cycle at 72° C. for 10min; followed by 4° C. soak. The PCR product was run on a 1% agarose gelto check the insert size. Those having the correct insert were used totransfect Spodoptera Frugiperda (Sf9) cells.

B. Transfection

Sf9 cells were seeded at 1×10⁶ cells per well in a 6-well plate andallowed to attach for 1 hour at 27° C. Five microliters of bacmid DNAwere diluted with 100 μl Sf-900 II SFM (Life Technologies). Twenty μl ofLipofectamine™ Reagent (Life Technologies, Cat. No. 18324-012) werediluted with 100 μSf-900 II SFM. The bacmid DNA and lipid solutions weregently mixed and incubated 30-45 minutes at room temperature. The mediafrom one well of cells was aspirated, the cells were washed 1× with 2 mlfresh Sf-900 II SFM media. Eight hundred microliters of Sf-900 II SFMwas added to the lipid-DNA mixture. The wash media was aspirated and theDNA-lipid mix added to the cells. The cells were incubated at 27° C.overnight. The DNA-lipid mix was aspirated and 2 ml of Sf-900 II mediawas added to each plate. The plates were incubated at 27° C., 90%humidity, for 96 hours after which the virus was harvested.

C. Amplification

Sf9 cells were seeded at 1×10⁶ cells per well in a 6-well plate. 50 μlof virus from the transfection plate were placed in the well and theplate was incubated at 27° C., 90% humidity, for 96 hours after whichthe virus was harvested.

Sf9 cells were grown in 50 ml Sf-900 II SFM in a 125 ml shake flask toan approximate density of 1×10⁶ cells/ml. They were then infected with100 μl of the viral stock from the above plate and incubated at 27° C.for 3 days after which time the virus was harvested.

Example 19 Purification of Zcyto18-Cee from Sf9 Cells

The following procedure was used for purifying zCyto 18 polypeptidescontaining C-terminal Glu-Glu (EE) tags (SEQ ID NO:14), that wereexpressed in baculovirus. Conditioned media from Sf9 cells expressingzCyto18-CEE (Example 18) was filtered using a 0.22 μm Steriflip™ filter(Millipore) and one Complete™ protease inhibitor cocktail tablet(Boehringer) was added for every 50 mL of media. Total target proteinconcentrations of the concentrated conditioned media were determined viaSDS-PAGE and Western blot analysis using an anti-EE antibody (producedin-house) followed by a secondary anti-mIg HRP conjugated antibody.

Batch purification was accomplished by adding 250 μl of Protein GSepharose® 4 Fast Flow (Pharmacia) which was treated with anti-EEantibody (Protein G Sepharose/anti-EE beads), to 40 mLs of Sf9conditioned media. To capture the ZCYTO18-CEE, the media-bead mixturewas rocked overnight at 4° C. The beads were spun out of the media at1000 RPM for 10 minutes in a Beckman GS6R centrifuge. The beads werewashed using the following scheme (centrifugation and aspiration stepswere done after each wash): 1× with 1 mL cell lysis buffer (150 mMSodium Chloride, 50 mM Tris pH 8.0, and 1% NP-40); 1× with 1 mL washbuffer (650 mM Sodium Chloride, 50 mM Tris pH 8.0, and 1% NP-40); 1×with 1 mL cell lysis buffer. The beads were then suspended in 500 μlcell lysis buffer and submitted for N-terminal sequencing.

Example 20 N-terminal Amino Acid Sequence Analysis

Standard automated N-terminal polypeptide sequencing (Edman degradation)was performed using reagents from Applied Biosystems. N-terminalsequence analysis was performed on a Model 494 Protein Sequencer System(Applied Biosystems, Inc., Foster City, Calif.). Data analysis wasperformed with Model 610A Data Analysis System for Protein Sequencing,version 2.1a (Applied Biosystems).

A purified human ZCYTO18-CEE sample was supplied as captured on ProteinG Sepharose/anti-EE beads (Example 19). The beads were placed inreducing SDS PAGE sample buffer and on a boiling water bath beforerunning on SDS PAGE, using a Novex SDS PAGE system (4-12% Bis-Tris MESNuPAGE; Invitrogen) as per manufacturer's instructions. The gel waselectrotransferred to a Novex PVDF membrane (Invitrogen), and Coomassieblue stained (Sigma, St. Louis, Mo.) using standard methods.Corresponding anti-EE Western blots were performed to identify theZCYTO18 band for N-terminal protein sequencing. The mouse anti-EE IgGHRP conjugated antibody used was produced in house.

N-terminal sequence analysis of the secreted ZCYTO18 polypeptideverified the predicted cleavage site of the signal sequence resulting ina mature start of the ZCYTO18 precursor sequence at 22 (Ala) as shown inSEQ ID NO:3.

Example 21 Construction of BaF3 Cells Expressing the CRF2-4 Receptor(BaF3/CRF2-4 Cells) and BaF3 Cells Expressing the CRF2-4 Receptor withthe Zcytor11 Receptor (BaF3/CRF2-4/Zcytor11 Cells)

BaF3 cells expressing the full-length CFR2-4 receptor were constructed,using 30 μg of a CFR2-4 expression vector, described below. The BaF3cells expressing the CFR2-4 receptor were designated as BaF3/CFR2-4.These cells were used as a control, and were further transfected withfull-length zcytor11 receptor (SEQ ID NO:18 and SEQ ID NO:19) (U.S. Pat.No. 5,965,704) and used to construct a screen for ZCYTO18 activity asdescribed below. This cell assay system can be used to assess ZCYTO18activity and readily screen for the activity of ZCYTO18 variants.

A. Construction of BaF3 Cells Expressing the CRF2-4 Receptor

The full-length cDNA sequence of CRF2-4 (Genbank Accession No. Z17227)was isolated from a Daudi cell line cDNA library, and then cloned intoan expression vector pZP7P using standard methods.

BaF3, an interleukin-3 (IL-3) dependent pre-lymphoid cell line derivedfrom murine bone marrow (Palacios and Steinmetz, Cell 41: 727-734, 1985;Mathey-Prevot et al., Mol. Cell. Biol. 6: 4133-4135, 1986), wasmaintained in complete media (RPMI medium (JRH Bioscience Inc., Lenexa,Kans.) supplemented with 10% heat-inactivated fetal calf serum, 2 ng/mlmurine IL-3 (mIL-3) (R & D, Minneapolis, Minn.), 2 mM L-glutaMax-1™(Gibco BRL), 1 mM Sodium Pyruvate (Gibco BRL), and PSN antibiotics(GIBCO BRL)). Prior to electroporation, CRF2-4/pZP7P was prepared andpurified using a Qiagen Maxi Prep kit (Qiagen) as per manufacturer'sinstructions. For electroporation, BaF3 cells were washed once inserum-free RPMI media and then resuspended in serum-free RPMI media at acell density of 10⁷ cells/ml. One ml of resuspended BaF3 cells was mixedwith 30 μg of the CRF2-4/pZP7P plasmid DNA and transferred to separatedisposable electroporation chambers (GIBCO BRL). Following a 15-minuteincubation at room temperature the cells were given two serial shocks(800 lFad/300 V.; 1180 lFad/300 V.) delivered by an electroporationapparatus (CELL-PORATOR™; GIBCO BRL). After a 5-minute recovery time,the electroporated cells were transferred to 50 ml of complete media andplaced in an incubator for 15-24 hours (37° C., 5% CO₂). The cells werethen spun down and resuspended in 50 ml of complete media containing 2μg/ml puromycin in a T-162 flask to isolate the puromycin-resistantpool. Pools of the transfected BaF3 cells, hereinafter calledBaF3/CRF2-4 cells, were assayed for signaling capability as describedbelow. Moreover these cells were further transfected with zcytor11receptor as described below.

B. Construction of BaF3 Cells Expressing CRF2-4 and Zcytor11 Receptors

BaF3/CRF2-4 cells expressing the full-length zcytor11 receptor wereconstructed as per Example 21A above, using 30 μg of an expressionvector containing zcytor11 cDNA (SEQ ID NO:18). Following recovery,transfectants were selected using 200 μg/ml zeocin and 2 μg/mlpuromycin. The BaF3/CRF2-4 cells expressing the zcytor11 receptor weredesignated as BaF3/CRF2-4/zcytor11 cells. These cells were used toscreen for ZCYTO18 activity (Example 22).

Example 22 Screening for Zcyto18 Activity Using BaF3/CRF2-4/Zcytor11Cells Using an Alamar Blue Proliferation Assay

A. Screening for Zcyto18 Activity Using BaF3/CRF2-4/Zcytor11 Cells Usingan Alamar Blue Proliferation Assay

Purified ZCYTO18-CEE (Example 9) was used to test for the presence ofproliferation activity as described below

BaF3/CRF2-4/zcytor11 cells were spun down and washed in the completemedia, described in Example 21A above, but without mIL-3 (hereinafterreferred to as “mIL-3 free media”). The cells were spun and washed 3times to ensure the removal of the mIL-3 Cells were then counted in ahemacytometer. Cells were plated in a 96-well format at 5000 cells perwell in a volume of 100 μl per well using the mIL-3 free media.

Proliferation of the BaF3/CRF2-4/zcytor11 cells was assessed usingZCYTO18-CEE protein diluted with mIL-3 free media to 50, 10, 2, 1, 0.5,0.25, 0.13, 0.06 ng/ml concentrations. 100 μl of the diluted protein wasadded to the BaF3/CRF2-4/zcytor11 cells. The total assay volume is 200μl. The assay plates were incubated at 37° C., 5% CO₂ for 3 days atwhich time Alamar Blue (Accumed, Chicago, Ill.) was added at 20 μl/well.Plates were again incubated at 37° C., 5% CO₂ for 24 hours. Alamar Bluegives a fluourometric readout based on number of live cells, and is thusa direct measurement of cell proliferation in comparison to a negativecontrol. Plates were again incubated at 37° C., 5% CO₂ for 24 hours.Plates were read on the Fmax™ plate reader (Molecular Devices Sunnyvale,Calif.) using the SoftMax™ Pro program, at wavelengths 544 (Excitation)and 590 (Emmission). Results confirmed the dose-dependent proliferativeresponse of the BaF3/CRF2-4/zcytor11 cells to ZCYTO18-CEE. The response,as measured, was approximately 15-fold over background at the high endof 50 ng/ml down to a 2-fold induction at the low end of 0.06 ng/ml. TheBaF3 wild type cells, and BaF3/CRF2-4 cells did not proliferate inresponse to ZCYTO18-CEE, showing that ZCYTO18 is specific for theCRF2-4/zcytor11 heterodimeric receptor.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

What is claimed is:
 1. A method of producing an antibody to apolypeptide comprising: inoculating an animal with a peptide consistingof amino acids 145 (Glu) to 150 (Lys) of SEQ ID NO:3; wherein thepolypeptide elicits an immune response in the animal to produce theantibody; and isolating the antibody from the animal.
 2. An isolatedantibody produced by the method of claim 1, which specifically binds toa polypeptide of SEQ ID NO:2 or SEQ ID NO:3.
 3. An isolated antibodythat specifically binds to an epitope of IL10-related T-cell derivedinducible factor (IL-TIF), wherein such epitope consists of amino acids145 (Glu) to 150 (Lys) of SEQ ID NO:3.
 4. The antibody of claim 3,wherein the antibody antagonizes IL-TIF activity.
 5. The antibody ofclaim 4, wherein the antibody is a monoclonal antibody.
 6. The antibodyof claim 4, wherein the antibody is an antibody fragment.
 7. Apharmaceutical composition comprising the antibody of claim
 4. 8. Thepharmaceutical composition of claim 7, wherein the antibody if amonoclonal antibody.
 9. The pharmaceutical composition of claim 7,wherein the antibody is an antibody fragment.